Cellobiohydrolase variants and polynucleotides encoding same

ABSTRACT

The present invention relates to cellobiohydrolase variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/773,715, filed Sep. 8, 2015, now U.S. Pat. No. 10,221,406 B2,which is a 35 U.S.C. § 371 national application of PCT/US2014/022068,filed Mar. 7, 2014, which claims priority from U.S. ProvisionalApplication No. 61/775,153, filed Mar. 8, 2013, the contents of whichare fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to cellobiohydrolase variants,polynucleotides encoding the variants, and methods of producing andusing the variants.

Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the lignocellulose is converted tofermentable sugars, e.g., glucose, the fermentable sugars can easily befermented by yeast into ethanol.

WO 2011/050037 discloses Thielavia terrestris cellobiohydrolase variantswith improved thermostability. WO 2011/050037 discloses Aspergillusfumigatus cellobiohydrolase variants with improved thermostability. WO2005/028636 discloses variants of Hypocrea jecorina Cel7Acellobiohydrolase I. WO 2005/001065 discloses variants of Humicolagrisea Cel7A cellobiohydrolase I, Hypocrea jecorina cellobiohydrolase I,and Scytalidium thermophilium cellobiohydrolase I. WO 2004/016760discloses variants of Hypocrea jecorina Cel7A cellobiohydrolase I. U.S.Pat. No. 7,375,197 discloses variants of Trichoderma reeseicellobiohydrolase I.

There is a need in the art for cellobiohydrolase variants with improvedproperties to increase the efficiency of the saccharification oflignocellulosic feedstocks.

The present invention provides cellobiohydrolase variants with increasedspecific performance, polynucleotides encoding the variants, and methodsof producing and using the variants.

SUMMARY OF THE INVENTION

The present invention relates to isolated cellobiohydrolase variants,comprising an alteration at one or more positions corresponding topositions 197, 198, 199, and 200 of the mature polypeptide of SEQ ID NO:2, wherein the alteration at the one or more positions corresponding topositions 197, 198, and 200 is a substitution and the alteration at theposition corresponding to position 199 is a deletion, and wherein thevariants have cellobiohydrolase activity.

The present invention also relates to isolated polynucleotides encodingthe variants; nucleic acid constructs, vectors, and host cellscomprising the polynucleotides; and methods of producing the variants.

The present invention also relates to processes for degrading acellulosic material, comprising: treating the cellulosic material withan enzyme composition in the presence of a cellobiohydrolase variant ofthe present invention. In one aspect, the processes further compriserecovering the degraded cellulosic material.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of acellobiohydrolase variant of the present invention; (b) fermenting thesaccharified cellulosic material with one or more (e.g., several)fermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme composition in thepresence of a cellobiohydrolase variant of the present invention. In oneaspect, the fermenting of the cellulosic material produces afermentation product. In another aspect, the processes further compriserecovering the fermentation product from the fermentation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows saccharide production from microcrystalline cellulose bythe Trichoderma reesei wild-type cellobiohydrolase I and thecellobiohydrolase I M6 variant thereof after 5 hours at pH 5 and 25° C.The results were corrected for background sugars determined in a controlsample.

FIG. 2 shows the effect of T. reesei cellobiohydrolase I M6 variant andT. reesei wild-type cellobiohydrolase I on hydrolysis of milled unwashedPCS by a cellulolytic enzyme composition at 24 hours.

FIG. 3 shows the effect of Trichoderma reesei cellobiohydrolase I M6variant and Trichoderma reesei wild-type cellobiohydrolase I onhydrolysis of milled unwashed pretreated corn stover (PCS) by acellulolytic enzyme composition at 48 hours.

FIG. 4 shows the effect of T. reesei cellobiohydrolase I M6 variant andT. reesei wild-type cellobiohydrolase I on hydrolysis of milled unwashedPCS by a cellulolytic enzyme composition at 72 hours.

FIG. 5 shows hydrolysis of microcrystalline cellulose by the T. reeseiwild-type cellobiohydrolase I and the T. reesei cellobiohydrolase IA199*, N198A, N200G, and N200W variants. Values are shown in mM releasedcellobiose after 1 hour at pH 5, at 25° C. and at 1100 rpm.

FIG. 6 shows hydrolysis of microcrystalline cellulose by the Rasamsoniaemersonii wild-type cellobiohydrolase I and R. emersoniicellobiohydrolase I PC1-146 variant. Values are shown in mM releasedcellobiose after 1 hour at pH 5, at 50° C. and at 1100 rpm.

FIG. 7 shows a comparison of the effect of the R. emersoniicellobiohydrolase I variant and R. emersonii wild-type cellobiohydrolaseI on the hydrolysis of unwashed PCS (8% total solids) by a cellulaseenzyme composition.

FIG. 8 shows a comparison of the effect of the R. emersoniicellobiohydrolase I PC1-146 variant and R. emersonii wild-typecellobiohydrolase I on the hydrolysis of unwashed PCS (20% total solids)by a cellulase enzyme composition.

FIG. 9 shows a comparison of the R. emersonii cellobiohydrolase IPC1-146 variant with the R. emersonii wild-type cellobiohydrolase Iduring hydrolysis.

FIG. 10 shows a comparison of the R. emersonii cellobiohydrolase IPC1-146 variant with the Rasamsonia emersonii wild-typecellobiohydrolase I during simultaneous saccharification andfermentation (SSF).

FIG. 11 shows a comparison of the hydrolysis of microcrystallinecellulose by the R. emersonii wild-type cellobiohydrolase I, R.emersonii PC1-147 fusion cellobiohydrolase I, and R. emersoniicellobiohydrolase I PC1-378 variant.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). Oneunit of acetylxylan esterase is defined as the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, J. Basic Microbiol.42: 55-66. One unit of beta-glucosidase is defined as 1.0 μmole ofp-nitrophenolate anion produced per minute at 25° C., pH 4.8 from 1 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention,beta-xylosidase activity is determined using 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20 at pH 5, 40° C. One unit of beta-xylosidaseis defined as 1.0 μmole of p-nitrophenolate anion produced per minute at40° C., pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100mM sodium citrate containing 0.01% TWEEN® 20.

Carbohydrate binding module: The term “carbohydrate binding module”means a domain within a carbohydrate-active enzyme that providescarbohydrate-binding activity (Boraston et al., 2004, Biochem. J. 383:769-781). A majority of known carbohydrate binding modules (CBMs) arecontiguous amino acid sequences with a discrete fold. The carbohydratebinding module (CBM) is typically found either at the N-terminal or atthe C-terminal extremity of an enzyme. The term “carbohydrate bindingmodule” is also used interchangedly herein with the term “carbohydratebinding domain”.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme. In one aspect,the catalytic domain is amino acids 1 to 429 of SEQ ID NO: 2. In anotheraspect, the catalytic domain is amino acids 1 to 437 of SEQ ID NO: 8. Inanother aspect, the catalytic domain is amino acids 1 to 440 of SEQ IDNO: 10. In another aspect, the catalytic domain is amino acids 1 to 437of SEQ ID NO: 12. In another aspect, the catalytic domain is amino acids1 to 437 of SEQ ID NO: 14. In another aspect, the catalytic domain isamino acids 1 to 438 of SEQ ID NO: 16. In another aspect, the catalyticdomain is amino acids 1 to 437 of SEQ ID NO: 18. In another aspect, thecatalytic domain is amino acids 1 to 430 of SEQ ID NO: 20. In anotheraspect, the catalytic domain is amino acids 1 to 433 of SEQ ID NO: 22.

Catalytic domain coding sequence: The term “catalytic domain codingsequence” means a polynucleotide that encodes a domain catalyzingcellobiohydrolase activity. In one aspect, the catalytic domain codingsequence is nucleotides 52 to 1469 of SEQ ID NO: 1. In another aspect,the catalytic domain coding sequence is nucleotides 52 to 1389 of SEQ IDNO: 3. In another aspect, the catalytic domain coding sequence isnucleotides 52 to 1389 of SEQ ID NO: 4. In another aspect, the catalyticdomain coding sequence is nucleotides 79 to 1389 of SEQ ID NO: 7. Inanother aspect, the catalytic domain coding sequence is nucleotides 52to 1371 of SEQ ID NO: 9. In another aspect, the catalytic domain codingsequence is nucleotides 55 to 1482 of SEQ ID NO: 11. In another aspect,the catalytic domain coding sequence is nucleotides 76 to 1386 of SEQ IDNO: 13. In another aspect, the catalytic domain is nucleotides 76 to1386 of SEQ ID NO: 15. In another aspect, the catalytic domain codingsequence is nucleotides 55 to 1504 of SEQ ID NO: 17. In another aspect,the catalytic domain coding sequence is nucleotides 61 to 1350 of SEQ IDNO: 19. In another aspect, the catalytic domain coding sequence isnucleotides 55 to 1353 of SEQ ID NO: 21.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing end(cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of thechain (Teeri, 1997, Trends in Biotechnology 15: 160-167; Teeri et al.,1998, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity isdetermined according to the procedures described by Lever et al., 1972,Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters,149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187:283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In thepresent invention, cellobiohydrolase activity is preferably determinedaccording to Examples 8 and 9 herein.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic enzyme activity include:(1) measuring the total cellulolytic enzyme activity, and (2) measuringthe individual cellulolytic enzyme activities (endoglucanases,cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al.,2006, Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman N21filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman N21filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in PCS (or otherpretreated cellulosic material) for 3-7 days at a suitable temperaturesuch as 25° C.-80° C., e.g., 30° C., 35° C., 40° C., 45° C., 50° C., 55°C., 60° C., 65° C., or 70° C., and a suitable pH such as 4-9, e.g., 5.0,5.5, 6.0, 6.5, or 7.0, compared to a control hydrolysis without additionof cellulolytic enzyme protein. Typical conditions are 1 ml reactions,washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodiumacetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours, sugaranalysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories,Inc., Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In one aspect, the cellulosicmaterial is any biomass material. In another aspect, the cellulosicmaterial is lignocellulose, which comprises cellulose, hemicelluloses,and lignin.

In an embodiment, the cellulosic material is agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, or wood (including forestryresidue).

In another embodiment, the cellulosic material is arundo, bagasse,bamboo, corn cob, corn fiber, corn stover, miscanthus, rice straw,switchgrass, or wheat straw.

In another embodiment, the cellulosic material is aspen, eucalyptus,fir, pine, poplar, spruce, or willow.

In another embodiment, the cellulosic material is algal cellulose,bacterial cellulose, cotton linter, filter paper, microcrystallinecellulose (e.g., AVICEL®), or phosphoric-acid treated cellulose.

In another embodiment, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Endoglucanase: The term “endoglucanase” means a4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3-1,4 glucans suchas cereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat, 1991, Biochem. J.280: 309-316, and Henrissat, and Bairoch, 1996, Biochem. J. 316:695-696. The GH61 polypeptides have recently been classified as lyticpolysaccharide monooxygenases (Quinlan et al., 2011, Proc. Natl. Acad.Sci. USA 208: 15079-15084; Phillips et al., 2011, ACS Chem. Biol. 6:1399-1406; Lin et al., 2012, Structure 20: 1051-1061) and are designated“Auxiliary Activity 9” or “AA9” polypeptides.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase (FAE) is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. For purposes of the present invention,feruloyl esterase activity is determined using 0.5 mMp-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. Oneunit of feruloyl esterase equals the amount of enzyme capable ofreleasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25° C.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment hascellobiohydrolase activity. In one aspect, a fragment contains at least420 amino acid residues, e.g., at least 445 amino acid residues or atleast 470 amino acid residues of the mature polypeptide of SEQ ID NO: 2or a variant thereof. In another aspect, a fragment contains at least430 amino acid residues, e.g., at least 455 amino acid residues or atleast 480 amino acid residues of the mature polypeptide of SEQ ID NO: 8or a variant thereof. In another aspect, a fragment contains at least380 amino acid residues, e.g., at least 400 amino acid residues or atleast 420 amino acid residues of the mature polypeptide of SEQ ID NO: 10or a variant thereof. In another aspect, a fragment contains at least380 amino acid residues, e.g., at least 400 amino acid residues or atleast 420 amino acid residues of the mature polypeptide of SEQ ID NO: 12ora variant thereof. In another aspect, a fragment contains at least 430amino acid residues, e.g., at least 455 amino acid residues or at least480 amino acid residues of the mature polypeptide of SEQ ID NO: 14 or avariant thereof. In another aspect, a fragment contains at least 430amino acid residues, e.g., at least 455 amino acid residues or at least480 amino acid residues of the mature polypeptide of SEQ ID NO: 16 or avariant thereof. In another aspect, a fragment contains at least 380amino acid residues, e.g., at least 400 amino acid residues or at least420 amino acid residues of the mature polypeptide of SEQ ID NO: 18 or avariant thereof. In another aspect, a fragment contains at least 370amino acid residues, e.g., at least 390 amino acid residues or at least410 amino acid residues of the mature polypeptide of SEQ ID NO: 20 or avariant thereof. In another aspect, a fragment contains at least 435amino acid residues, e.g., at least 460 amino acid residues or at least485 amino acid residues of the mature polypeptide of SEQ ID NO: 22 or avariant thereof.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom andShoham, 2003, Current Opinion In Microbiology 6(3): 219-228).Hemicellulases are key components in the degradation of plant biomass.Examples of hemicellulases include, but are not limited to, anacetylmannan esterase, an acetylxylan esterase, an arabinanase, anarabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. The substrates for theseenzymes, hemicelluloses, are a heterogeneous group of branched andlinear polysaccharides that are bound via hydrogen bonds to thecellulose microfibrils in the plant cell wall, crosslinking them into arobust network. Hemicelluloses are also covalently attached to lignin,forming together with cellulose a highly complex structure. The variablestructure and organization of hemicelluloses require the concertedaction of many enzymes for its complete degradation. The catalyticmodules of hemicellulases are either glycoside hydrolases (GHs) thathydrolyze glycosidic bonds, or carbohydrate esterases (CEs), whichhydrolyze ester linkages of acetate or ferulic acid side groups. Thesecatalytic modules, based on homology of their primary sequence, can beassigned into GH and CE families. Some families, with an overall similarfold, can be further grouped into clans, marked alphabetically (e.g.,GH-A). A most informative and updated classification of these and othercarbohydrate active enzymes is available in the Carbohydrate-ActiveEnzymes (CAZy) database. Hemicellulolytic enzyme activities can bemeasured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59:1739-1752, at a suitable temperature such as 40° C.-80° C., e.g., 50°C., 55° C., 60° C., 65° C., or 70° C., and a suitable pH such as 4-9,e.g., 5.0, 5.5, 6.0, 6.5, or 7.0.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 0.2×SSC, 0.2% SDSat 65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parent. Suchan improved property is preferably increased specific performance.

Increased specific performance: The term “increased specificperformance” by a variant of the present invention means improvedconversion of a cellulosic material to a product, as compared to thesame level of conversion by the parent. Increased specific performanceis determined per unit protein (e.g., mg protein, or μmole protein). Theincreased specific performance of the variant relative to the parent canbe assessed, for example, under one or more (e.g., several) conditionsof pH, temperature, and substrate concentration. In one aspect, theproduct is glucose. In another aspect, the product is cellobiose. Inanother aspect, the product is glucose+cellobiose.

In one aspect, the condition is pH. For example, the pH can be any pH inthe range of 3 to 7, e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or7.0 (or in between). Any suitable buffer for achieving the desired pHcan be used.

In another aspect, the condition is temperature. For example, thetemperature can be any temperature in the range of 25° C. to 90° C.,e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90° C. (orin between).

In another aspect, the condition is substrate concentration. Anycellulosic material defined herein can be used as the substrate. In oneaspect, the substrate concentration is measured as the dry solidscontent. The dry solids content is preferably in the range of about 1 toabout 50 wt %), e.g., about 5 to about 45 wt %), about 10 to about 40 wt%), or about 20 to about 30 wt In another aspect, the substrateconcentration is measured as the insoluble glucan content. The insolubleglucan content is preferably in the range of about 2.5 to about 25 wte.g., about 5 to about 20 wt % or about 10 to about 15 wt %.

In another aspect, a combination of two or more (e.g., several) of theabove conditions are used to determine the increased specificperformance of the variant relative to the parent, such as anytemperature in the range of 25° C. to 90° C., e.g., 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, or 90° C. (or in between) at a pH in therange of 3 to 7, e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0(or in between).

The increased specific performance of the variant relative to the parentcan be determined using any enzyme assay known in the art forcellobiohydrolases as described herein. Alternatively, the increasedspecific performance of the variant relative to the parent can bedetermined using the assays described in Examples 8 and 9.

In another aspect, the specific performance of the variant is at least1.01-fold, e.g., at least 1.02-fold, at least 1.03-fold, at least1.04-fold, at least 1.05-fold, at least 1.06-fold, at least 1.07-fold,at least 1.08-fold, at least 1.09-fold, at least 1.1-fold, at least1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, atleast 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold,at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 5-fold, atleast 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, andat least 50-fold higher than the specific performance of the parent.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance).

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 0.2×SSC, 0.2% SDSat 50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 497 of SEQ ID NO: 2 based on the SignalP3.0 program (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) thatpredicts amino acids −1 to −17 of SEQ ID NO: 2 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 1 to 506 of SEQ IDNO: 8 (P3EX) based on the SignalP 3.0 program that predicts amino acids−1 to −26 of SEQ ID NO: 8 are a signal peptide. In another aspect, themature polypeptide is amino acids 1 to 440 of SEQ ID NO: 10 (P57J) basedon the SignalP 3.0 program that predicts amino acids −1 to −17 of SEQ IDNO: 10 are a signal peptide. In another aspect, the mature polypeptideis amino acids 1 to 437 of SEQ ID NO: 12 (P82PH) based on the SignalP3.0 program that predicts amino acids −1 to −18 of SEQ ID NO: 12 are asignal peptide. In another aspect, the mature polypeptide is amino acids1 to 507 of SEQ ID NO: 14 (P23YSY) based on the SignalP 3.0 program thatpredicts amino acids −1 to −25 of SEQ ID NO: 14 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 1 to 507 of SEQ IDNO: 16 (P23YSX) based on the SignalP 3.0 program that predicts aminoacids −1 to −25 of SEQ ID NO: 16 are a signal peptide. In anotheraspect, the mature polypeptide is amino acids 1 to 437 of SEQ ID NO: 18(P247B5) based on the SignalP 3.0 program that predicts amino acids −1to −18 of SEQ ID NO: 18 are a signal peptide. In another aspect, themature polypeptide is amino acids 1 to 430 of SEQ ID NO: 20 (P66Z) basedon the SignalP 3.0 program that predicts amino acids −1 to −20 of SEQ IDNO: 20 are a signal peptide. In another aspect, the mature polypeptideis amino acids 1 to 511 of SEQ ID NO: 22 (P57G) based on the SignalP 3.0program that predicts amino acids −1 to −18 of SEQ ID NO: 22 are asignal peptide. It is known in the art that a host cell may produce amixture of two of more different mature polypeptides (i.e., with adifferent C-terminal and/or N-terminal amino acid) expressed by the samepolynucleotide. It is also known in the art that different host cellsmay process polypeptides differently, and thus, one host cell expressinga polynucleotide may produce a different mature polypeptide (e.g.,having a different C-terminal and/or N-terminal amino acid) as comparedto another host cell expressing the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellobiohydrolase activity. In one aspect, the mature polypeptidecoding sequence is nucleotides 52 to 1673 of SEQ ID NO: 1 (without thestop codon) based on SignalP 3.0 program (Bendtsen et al., 2004, supra)that predicts nucleotides 1 to 51 of SEQ ID NO: 1 encode a signalpeptide. In another aspect, the mature polypeptide coding sequence isnucleotides 52 to 1542 of SEQ ID NO: 3 (without the stop codon) based onSignalP 3.0 program that predicts nucleotides 1 to 51 of SEQ ID NO: 3encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 52 to 1542 of SEQ ID NO: 4 (without thestop codon) based on SignalP 3.0 program that predicts nucleotides 1 to51 of SEQ ID NO: 4 encode a signal peptide. In another aspect, themature polypeptide coding sequence is nucleotides 79 to 1596 of SEQ IDNO: 7 (D1R9) (without the stop codon) based on SignalP 3.0 program thatpredicts nucleotides 1 to 78 of SEQ ID NO: 7 encode a signal peptide. Inanother aspect, the mature polypeptide coding sequence is nucleotides 52to 1371 of SEQ ID NO: 9 (D3FQ) (without the stop codon) based on SignalP3.0 program that predicts nucleotides 1 to 51 of SEQ ID NO: 9 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is nucleotides 55 to 1482 of SEQ ID NO: 11 (D23Y2) (without thestop codon) based on SignalP 3.0 program that predicts nucleotides 1 to54 of SEQ ID NO: 11 encode a signal peptide. In another aspect, themature polypeptide coding sequence is nucleotides 76 to 1596 of SEQ IDNO: 13 (D72PP3) (without the stop codon) based on SignalP 3.0 programthat predicts nucleotides 1 to 75 of SEQ ID NO: 13 encode a signalpeptide. In another aspect, the mature polypeptide coding sequence isnucleotides 76 to 1596 of SEQ ID NO: 15 (D72PP2) (without the stopcodon) based on SignalP 3.0 program that predicts nucleotides 1 to 75 ofSEQ ID NO: 15 encode a signal peptide. In another aspect, the maturepolypeptide coding sequence is nucleotides 55 to 1504 of SEQ ID NO: 17(D82ACF) (without the stop codon) based on SignalP 3.0 program thatpredicts nucleotides 1 to 54 of SEQ ID NO: 17 encode a signal peptide.In another aspect, the mature polypeptide coding sequence is nucleotides61 to 1350 of SEQ ID NO: 19 (D6CT) (without the stop codon) based onSignalP 3.0 program that predicts nucleotides 1 to 60 of SEQ ID NO: 19encode a signal peptide. In another aspect, the mature polypeptidecoding sequence is nucleotides 55 to 1587 of SEQ ID NO: 21 (D3FP)(without the stop codon) based on SignalP 3.0 program that predictsnucleotides 1 to 54 of SEQ ID NO: 21 encode a signal peptide.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 60° C.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence. Parent or parentcellobiohydrolase: The term “parent” or “parent cellobiohydrolase” meansa polypeptide having cellobiohydrolase activity to which an alteration,i.e., a substitution, insertion, and/or deletion, at one or more (e.g.,several) positions, is made to produce an enzyme variant of the presentinvention. The parent may be a naturally occurring (wild-type)polypeptide or a variant or fragment thereof.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide or variant thereof that catalyzes the enhancement of thehydrolysis of a cellulosic material by enzyme having cellulolyticactivity, i.e., a cellulase. For purposes of the present invention,cellulolytic enhancing activity is determined by measuring the increasein reducing sugars or the increase of the total of cellobiose andglucose from the hydrolysis of a cellulosic material by cellulolyticenzyme under the following conditions: 1-50 mg of total protein/g ofcellulose in pretreated corn stover (PCS), wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of a GH61 polypeptide or variant thereof for 1-7 days at asuitable temperature, such as 25° C.-80° C., e.g., 30° C., 35° C., 40°C., 45° C., 50° C., 55° C., 60° C., 65° C., or 70° C., and a suitablepH, such as 4-9, e.g., 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5,compared to a control hydrolysis with equal total protein loadingwithout cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS) In one aspect, GH61 polypeptide enhancingactivity is determined using a mixture of CELLUCLAST® 1.5 L (NovozymesA/S, Bagsvrd, Denmark) in the presence of 2-3% of total protein weightAspergillus oryzae beta-glucosidase (recombinantly produced inAspergillus oryzae according to WO 02/095014) or 2-3% of total proteinweight Aspergillus fumigatus beta-glucosidase (recombinantly produced inAspergillus oryzae as described in WO 02/095014) of cellulase proteinloading is used as the source of the cellulolytic activity.

Another assay for determining the cellulolytic enhancing activity of aGH61 polypeptide or variant thereof is to incubate the GH61 polypeptideor variant with 0.5% phosphoric acid swollen cellulose (PASC), 100 mMsodium acetate pH 5, 1 mM MnSO₄, 0.1% gallic acid, 0.025 mg/ml ofAspergillus fumigatus beta-glucosidase, and 0.01% TRITON® X100(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) for 24-96 hoursat 40° C. followed by determination of the glucose released from thePASC.

The GH61 polypeptides or variants thereof having cellulolytic enhancingactivity enhance the hydrolysis of a cellulosic material catalyzed byenzyme having cellulolytic activity by reducing the amount ofcellulolytic enzyme required to reach the same degree of hydrolysispreferably at least 1.01-fold, e.g., at least 1.05-fold, at least1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, atleast 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or atleast 20-fold.

Pretreated corn stover: The term “Pretreated Corn Stover” or “PCS” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, neutral pretreatment,or any pretreatment known in the art.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used area gap open penalty of 10, a gap extension penalty of 0.5, and theEBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the -nobriefoption) is used as the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are a gap open penalty of10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version ofNCBI NUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows: (IdenticalDeoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps inAlignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having cellobiohydrolase activity. In one aspect, a subsequencecontains at least 1400 nucleotides, e.g., at least 1475 nucleotides orat least 1550 nucleotides of the mature polypeptide coding sequence ofSEQ ID NO: 1 or a mutant thereof. In another aspect, a subsequencecontains at least 1260 nucleotides, e.g., at least 1335 nucleotides orat least 1410 nucleotides of the mature polypeptide coding sequence ofSEQ ID NO: 3 or a mutant thereof. In another aspect, a subsequencecontains at least 1260 nucleotides, e.g., at least 1335 nucleotides orat least 1410 nucleotides of the mature polypeptide coding sequence ofSEQ ID NO: 4 or a mutant thereof. In another aspect, a fragment containsat least 1290 nucleotides, e.g., at least 1365 nucleotides or at least1440 nucleotides of the mature polypeptide of SEQ ID NO: 7 ora mutantthereof. In another aspect, a fragment contains at least 1140nucleotides, e.g., at least 1200 nucleotides or at least 1260nucleotides of the mature polypeptide of SEQ ID NO: 9 ora mutantthereof. In another aspect, a fragment contains at least 1140nucleotides, e.g., at least 1200 nucleotides or at least 1260nucleotides of the mature polypeptide of SEQ ID NO: 11 ora mutantthereof. In another aspect, a fragment contains at least 1290nucleotides, e.g., at least 1365 nucleotides or at least 1440nucleotides of the mature polypeptide of SEQ ID NO: 13 ora mutantthereof. In another aspect, a fragment contains at least 1290nucleotides, e.g., at least 1365 nucleotides or at least 1440nucleotides of the mature polypeptide of SEQ ID NO: 15 ora mutantthereof. In another aspect, a fragment contains at least 1140nucleotides, e.g., at least 1200 nucleotides or at least 1260nucleotides of the mature polypeptide of SEQ ID NO: 17 ora mutantthereof. In another aspect, a fragment contains at least 1110nucleotides, e.g., at least 1170 nucleotides or at least 1230nucleotides of the mature polypeptide of SEQ ID NO: 19 ora mutantthereof. In another aspect, a fragment contains at least 1305nucleotides, e.g., at least 1380 nucleotides or at least 1455nucleotides of the mature polypeptide of SEQ ID NO: 21 or a mutantthereof.

Variant: The term “variant” means a polypeptide having cellobiohydrolaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition. The variants of the present invention have a specificperformance which is at least 1.01-fold higher than the specificperformance of the parent.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS at 45° C.

Wild-type cellobiohydrolase: The term “wild-type” cellobiohydrolasemeans a cellobiohydrolase naturally produced by a microorganism, such asa bacterium, yeast, or filamentous fungus found in nature.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the processes of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, 2006, Journal of the Science of Food and Agriculture 86(11):1636-1647; Spanikova and Biely, 2006, FEBS Letters 580(19): 4597-4601;Herrimann et al., 1997, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. A common total xylanolytic activity assay is based onproduction of reducing sugars from polymeric 4-O-methyl glucuronoxylanas described in Bailey et al., 1992, Interlaboratory testing of methodsfor assay of xylanase activity, Journal of Biotechnology 23(3): 257-270.Xylanase activity can also be determined with 0.2% AZCL-arabinoxylan assubstrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 μmole of azurineproduced per minute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan assubstrate in 200 mM sodium phosphate pH 6.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37° C. One unit ofxylanase activity is defined as 1.0 μmole of azurine produced per minuteat 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mMsodium phosphate pH 6.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 2 is used to determine the corresponding amino acidresidue in another cellobiohydrolase. The amino acid sequence of anothercellobiohydrolase is aligned with the mature polypeptide disclosed inSEQ ID NO: 2, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the mature polypeptidedisclosed in SEQ ID NO: 2 is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277), preferably version 5.0.0 or later. Theparameters used are a gap open penalty of 10, a gap extension penalty of0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.Numbering of the amino acid positions is based on the full-lengthpolypeptide (e.g., including the signal peptide) of SEQ ID NO: 2 whereinposition −17 is the first amino acid of the signal peptide (i.e., Met)and position 1 is Gln of SEQ ID NO: 2. For example, the positioncorresponding to position 197 of the Trichoderma reeseicellobiohydrolase I (SEQ ID NO: 2) is position 194 of the Rasamsoniaemersonii cellobiohydrolase I, and position 200 of the Trichodermareesei cellobiohydrolase I is position 197 of the Rasamsonia emersoniicellobiohydrolase I.

Identification of the corresponding amino acid residue in anothercellobiohydrolase can be determined by an alignment of multiplepolypeptide sequences using several computer programs including, but notlimited to, MUSCLE (multiple sequence comparison by log-expectation;version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, NucleicAcids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research33: 511-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh etal., 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010,Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When another cellobiohydrolase has diverged from the mature polypeptideof SEQ ID NO: 2 such that traditional sequence-based comparison fails todetect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol.295: 613-615), other pairwise sequence comparison algorithms can beused. Greater sensitivity in sequence-based searching can be attainedusing search programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the cellobiohydrolase variants of the present invention,the nomenclature described below is adapted for ease of reference. Theaccepted IUPAC single letter or three letter amino acid abbreviation isemployed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. Multiple mutations areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G-K-A

Multiple alterations. Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.

Different alterations. Where different alterations can be introduced ata position, the different alterations are separated by a comma, e.g.,“Arg170Tyr,Glu” represents a substitution of arginine at position 170with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants: “Tyr167Gly+Arg170Gly”,“Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated cellobiohydrolase variants,comprising an alteration at one or more positions corresponding topositions 197, 198, 199, and 200 of the mature polypeptide of SEQ ID NO:2, wherein the alteration at the one or more positions corresponding topositions 197, 198, and 200 is a substitution and at the positioncorresponding to position 199 is a deletion, and wherein the variantshave cellobiohydrolase activity.

Variants

In an embodiment, the variant has a sequence identity of at least 60%,e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%, tothe amino acid sequence of the parent cellobiohydrolase or the maturepolypeptide thereof.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, sequence identity to themature polypeptide of SEQ ID NO: 2.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, sequence identity to themature polypeptide of SEQ ID NO: 8.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, sequence identity to themature polypeptide of SEQ ID NO: 10.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, sequence identity to themature polypeptide of SEQ ID NO: 12.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, sequence identity to themature polypeptide of SEQ ID NO: 14.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, sequence identity to themature polypeptide of SEQ ID NO: 16.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, sequence identity to themature polypeptide of SEQ ID NO: 18.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, sequence identity to themature polypeptide of SEQ ID NO: 20.

In another embodiment, the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, sequence identity to themature polypeptide of SEQ ID NO: 22.

In one aspect, the number of alterations in the variants of the presentinvention is 1-4, e.g., 1, 2, 3, or 4 alterations. In another aspect,the number of substitutions in the variants of the present invention is1-3, e.g., 1, 2, or 3 substitutions. In another aspect, the number ofdeletions in the variants of the present invention is 1 deletion.

In another aspect, a variant comprises an alteration at one or morepositions corresponding to positions 197, 198, 199, and 200 of themature polypeptide of SEQ ID NO: 2, wherein the alteration at the one ormore positions corresponding to positions 197, 198, and 200 is asubstitution and the alteration at the position corresponding toposition 199 is a deletion. In another aspect, a variant comprises analteration at two positions corresponding to any of positions 197, 198,199, and 200 of the mature polypeptide of SEQ ID NO: 2, wherein thealteration at the one or more positions corresponding to positions 197,198, and 200 is a substitution and the alteration at the positioncorresponding to position 199 is a deletion. In another aspect, avariant comprises an alteration at three positions corresponding to anyof positions 197, 198, 199, and 200 of the mature polypeptide of SEQ IDNO: 2, wherein the alteration at the one or more positions correspondingto positions 197, 198, and 200 is a substitution and the alteration atthe position corresponding to position 199 is a deletion. In anotheraspect, a variant comprises a substitution at each positioncorresponding to positions 197, 198, and 200 and a deletion at aposition corresponding to position 199.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 197. In another aspect, theamino acid at a position corresponding to position 197 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala. In anotheraspect, the variant comprises or consists of the substitution N197A ofthe mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 198. In another aspect, theamino acid at a position corresponding to position 198 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala. In anotheraspect, the variant comprises or consists of the substitution N198A ofthe mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of a deletion at aposition corresponding to position 199. In another aspect, the aminoacid at a position corresponding to position 199 is Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val, preferably Ala. In another aspect, the variant comprises orconsists of the deletion A199* of the mature polypeptide of SEQ ID NO:2.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 200. In another aspect, theamino acid at a position corresponding to position 200 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably with Ala, Gly, or Trp.In another aspect, the variant comprises or consists of the substitutionN200A,G,W of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of an alteration atpositions corresponding to positions 197 and 198, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 197 and 199, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 197 and 200, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 198 and 199, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 198 and 200, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 199 and 200, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 197, 198, and 199, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 197, 198, and 200, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 197, 199, and 200, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 198, 199, and 200, such as thosedescribed above.

In another aspect, the variant comprises or consists of alterations atpositions corresponding to positions 197, 198, 199, and 200, such asthose described above. In another aspect, the variant comprises orconsists of one or more alterations selected from the group consistingof N197A, N198A, A199*, and N200A,G,W.

In another aspect, the variant comprises or consists of the alterationsN197A+N198A of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the alterationsN197A+A199* of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the alterationsN197A+N200A,G,W of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the alterationsN198A+A199* of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the alterationsN198A+N200A,G,W of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the alterationsA199*+N200A,G,W of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the alterationsN197A+N198A+A199* of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the alterationsN197A+N198A+N200A,G,W of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the alterationsN197A+A199*+N200A,G,W of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the alterationsN198A+A199*+N200A,G,W of the mature polypeptide of SEQ ID NO: 2.

In another aspect, the variant comprises or consists of the alterationsN197A+N198A+A199*+N200A,G,W of the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the variant comprises or consists of SEQ ID NO: 6 orthe mature polypeptide thereof.

In another embodiment, the variant comprises or consists of SEQ ID NO:45 or the mature polypeptide thereof.

In another embodiment, the variant comprises or consists of SEQ ID NO:47 or the mature polypeptide thereof.

In another embodiment, the variant comprises or consists of SEQ ID NO:49 or the mature polypeptide thereof.

In another embodiment, the variant comprises or consists of SEQ ID NO:51 or the mature polypeptide thereof.

In another embodiment, the variant comprises or consists of SEQ ID NO:66 or the mature polypeptide thereof.

In another embodiment, the variant comprises or consists of SEQ ID NO:76 or the mature polypeptide thereof.

The variants may further comprise one or more additional alterations,e.g., substitutions, insertions, or deletions at one or more (e.g.,several) other positions.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

The variants may further or even further comprise one or more (e.g.,several) substitutions at positions corresponding to positions disclosedin WO 2011/050037, WO 2011/050037, WO 2005/02863, WO 2005/001065, WO2004/016760, and U.S. Pat. No. 7,375,197, which are incorporated hereinin their entireties.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for cellobiohydrolase activity to identify aminoacid residues that are critical to the activity of the molecule. Seealso, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity ofessential amino acids can also be inferred from an alignment with arelated polypeptide.

The variants may consist of 370 to 507 amino acids, e.g., 370 to 380,380 to 390, 390 to 400, 400 to 410, 410 to 420, 420 to 430, 430 to 440,450 to 460, 460 to 470, 470 to 480, 480 to 490, 490 to 500, or 500 to507 amino acids.

In each of the embodiments described above, a variant of the presentinvention may be a hybrid polypeptide (chimera) in which a region of thevariant is replaced with a region of another polypeptide. In one aspect,the region is a carbohydrate binding domain. The carbohydrate bindingdomain of a variant may be replaced with another (heterologous)carbohydrate binding domain.

In each of the embodiments described above, a variant of the presentinvention may be a fusion polypeptide or cleavable fusion polypeptide inwhich another polypeptide is fused at the N-terminus or the C-terminusof the variant. In one aspect, the other polypeptide is a carbohydratebinding domain. The catalytic domain of a variant of the presentinvention without a carbohydrate binding domain may be fused to acarbohydrate binding domain. A fusion polypeptide is produced by fusinga polynucleotide encoding another polypeptide to a polynucleotideencoding a variant of the present invention. Techniques for producingfusion polypeptides are known in the art, and include ligating thecoding sequences encoding the polypeptides so that they are in frame andexpression of the fusion polypeptide is under control of the samepromoter(s) and terminator. Fusion polypeptides may also be constructedusing intein technology in which fusion polypeptides are createdpost-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawsonet al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

In one embodiment, the variant is a hybrid or chimeric polypeptide inwhich the carbohydrate binding domain of the variant is replaced with adifferent carbohydrate binding domain. In another embodiment, thevariant is a fusion protein in which a heterologous carbohydrate bindingdomain is fused to the variant. In one aspect, the carbohydrate bindingdomain is fused to the N-terminus of the variant. In another aspect, thecarbohydrate binding domain is fused to the C-terminus of the variant.

In an embodiment, the variant has increased specific performancecompared to the parent enzyme.

Parent Cellobiohydrolases

The parent cellobiohydrolase may be any cellobiohydrolase I.

In one embodiment, the parent cellobiohydrolase may be (a) a polypeptidehaving at least 60% sequence identity to the mature polypeptide of SEQID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizesunder low stringency conditions with the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4; or thefull-length complement thereof; or (c) a polypeptide encoded by apolynucleotide having at least 60% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO:4.

In another embodiment, the parent cellobiohydrolase may also be (a) apolypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 8; (b) a polypeptide encoded by apolynucleotide that hybridizes under low stringency conditions with themature polypeptide coding sequence of SEQ ID NO: 7; or the full-lengthcomplement thereof; or (c) a polypeptide encoded by a polynucleotidehaving at least 60% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 7.

In another embodiment, the parent cellobiohydrolase may also be (a) apolypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 10; (b) a polypeptide encoded by apolynucleotide that hybridizes under low stringency conditions with themature polypeptide coding sequence of SEQ ID NO: 9 or the full-lengthcomplement thereof; or (c) a polypeptide encoded by a polynucleotidehaving at least 60% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 9.

In another embodiment, the parent cellobiohydrolase may also be (a) apolypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 12; (b) a polypeptide encoded by apolynucleotide that hybridizes under low stringency conditions with themature polypeptide coding sequence of SEQ ID NO: 11 or the full-lengthcomplement thereof; or (c) a polypeptide encoded by a polynucleotidehaving at least 60% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 11.

In another embodiment, the parent cellobiohydrolase may also be (a) apolypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 14; (b) a polypeptide encoded by apolynucleotide that hybridizes under low stringency conditions with themature polypeptide coding sequence of SEQ ID NO: 13 or the full-lengthcomplement thereof; or (c) a polypeptide encoded by a polynucleotidehaving at least 60% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 13.

In another embodiment, the parent cellobiohydrolase may also be (a) apolypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 16; (b) a polypeptide encoded by apolynucleotide that hybridizes under low stringency conditions with themature polypeptide coding sequence of SEQ ID NO: 15 or the full-lengthcomplement thereof; or (c) a polypeptide encoded by a polynucleotidehaving at least 60% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 15.

In another embodiment, the parent cellobiohydrolase may also be (a) apolypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 18; (b) a polypeptide encoded by apolynucleotide that hybridizes under low stringency conditions with themature polypeptide coding sequence of SEQ ID NO: 17 or the full-lengthcomplement thereof; or (c) a polypeptide encoded by a polynucleotidehaving at least 60% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 17.

In another embodiment, the parent cellobiohydrolase may also be (a) apolypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 20; (b) a polypeptide encoded by apolynucleotide that hybridizes under low stringency conditions with themature polypeptide coding sequence of SEQ ID NO: 19 or the full-lengthcomplement thereof; or (c) a polypeptide encoded by a polynucleotidehaving at least 60% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 19.

In another embodiment, the parent cellobiohydrolase may also be (a) apolypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 22; (b) a polypeptide encoded by apolynucleotide that hybridizes under low stringency conditions with themature polypeptide coding sequence of SEQ ID NO: 21 or the full-lengthcomplement thereof; or (c) a polypeptide encoded by a polynucleotidehaving at least 60% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 21.

In one aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 2 of at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, which have cellobiohydrolase activity.In another aspect, the amino acid sequence of the parent differs by upto 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from themature polypeptide of SEQ ID NO: 2.

In another aspect, the parent has a sequence identity to the maturepolypeptide of

SEQ ID NO: 8 of at least 60%, e.g., at least 65%, at least 70%, at least75%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%, which have cellobiohydrolase activity. In another aspect,the amino acid sequence of the parent differs by up to 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide ofSEQ ID NO: 8.

In another aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 10 of at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, which have cellobiohydrolase activity.In another aspect, the amino acid sequence of the parent differs by upto 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from themature polypeptide of SEQ ID NO: 10.

In another aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 12 of at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, which have cellobiohydrolase activity.In another aspect, the amino acid sequence of the parent differs by upto 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from themature polypeptide of SEQ ID NO: 12.

In another aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 14 of at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, which have cellobiohydrolase activity.In another aspect, the amino acid sequence of the parent differs by upto 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from themature polypeptide of SEQ ID NO: 14.

In another aspect, the parent has a sequence identity to the maturepolypeptide of

SEQ ID NO: 16 of at least 60%, e.g., at least 65%, at least 70%, atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which have cellobiohydrolase activity. In anotheraspect, the amino acid sequence of the parent differs by up to 10 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the maturepolypeptide of SEQ ID NO: 16.

In another aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 18 of at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, which have cellobiohydrolase activity.In another aspect, the amino acid sequence of the parent differs by upto 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from themature polypeptide of SEQ ID NO: 18.

In another aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 20 of at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, which have cellobiohydrolase activity.In another aspect, the amino acid sequence of the parent differs by upto 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from themature polypeptide of SEQ ID NO: 20.

In another aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 22 of at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, which have cellobiohydrolase activity.In another aspect, the amino acid sequence of the parent differs by upto 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from themature polypeptide of SEQ ID NO: 22.

In another aspect, the parent comprises or consists of the amino acidsequence of

SEQ ID NO: 2. In another aspect, the parent comprises or consists of themature polypeptide of SEQ ID NO: 2. In another aspect, the parentcomprises or consists of amino acids 1 to 497 of SEQ ID NO: 2.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 8. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 8. In another aspect,the parent comprises or consists of amino acids 1 to 506 of SEQ ID NO:8.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 10. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 10. In another aspect,the parent comprises or consists of amino acids 1 to 437 of SEQ ID NO:10.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 12. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 12. In another aspect,the parent comprises or consists of amino acids 1 to 437 of SEQ ID NO:12.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 14. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 14. In another aspect,the parent comprises or consists of amino acids 1 to 507 of SEQ ID NO:14.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 16. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 16. In another aspect,the parent comprises or consists of amino acids 1 to 507 of SEQ ID NO:16.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 18. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 18. In another aspect,the parent comprises or consists of amino acids 1 to 437 of SEQ ID NO:18.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 20. In another aspect, the parent comprises orconsists of the mature polypeptide of SEQ ID NO: 20. In another aspect,the parent comprises or consists of amino acids 1 to 430 of SEQ ID NO:20.

In another aspect, the parent comprises or consists of the amino acidsequence of

SEQ ID NO: 22. In another aspect, the parent comprises or consists ofthe mature polypeptide of SEQ ID NO: 22. In another aspect, the parentcomprises or consists of amino acids 1 to 511 of SEQ ID NO: 22.

In another aspect, the parent is a fragment of the mature polypeptide ofSEQ ID NO: 2 containing at least 420 amino acid residues, e.g., at least445 amino acid residues or at least 470 amino acid residues.

In another aspect, the parent is a fragment of the mature polypeptide ofSEQ ID NO: 8 containing at 430 amino acid residues, e.g., at least 455amino acid residues or at least 480 amino acid residues.

In another aspect, the parent is a fragment of the mature polypeptide ofSEQ ID NO: 10 containing at least 380 amino acid residues, e.g., atleast 400 amino acid residues or at least 420 amino acid residues.

In another aspect, the parent is a fragment of the mature polypeptide ofSEQ ID NO: 12 containing at least 380 amino acid residues, e.g., atleast 400 amino acid residues or at least 420 amino acid residues.

In another aspect, the parent is a fragment of the mature polypeptide ofSEQ ID NO: 14 containing at least 430 amino acid residues, e.g., atleast 455 amino acid residues or at least 480 amino acid residues.

In another aspect, the parent is a fragment of the mature polypeptide ofSEQ ID NO: 16 containing at least 430 amino acid residues, e.g., atleast 455 amino acid residues or at least 480 amino acid residues.

In another aspect, the parent is a fragment of the mature polypeptide ofSEQ ID NO: 18 containing at least 380 amino acid residues, e.g., atleast 400 amino acid residues or at least 420 amino acid residues.

In another aspect, the parent is a fragment of the mature polypeptide ofSEQ ID NO: 20 containing at least 370 amino acid residues, e.g., atleast 390 amino acid residues or at least 410 amino acid residues.

In another aspect, the parent is a fragment of the mature polypeptide ofSEQ ID NO: 22 containing at least 435 amino acid residues, e.g., atleast 460 amino acid residues or at least 485 amino acid residues.

In another aspect, the parent is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with the mature polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, or SEQ ID NO: 4; or the full-length complement thereof(Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y.).

In another aspect, the parent is encoded by a polynucleotide thathybridizes under low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 7; or the full-lengthcomplement thereof (Sambrook et al., 1989, supra).

In another aspect, the parent is encoded by a polynucleotide thathybridizes under low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 9; or the full-lengthcomplement thereof (Sambrook et al., 1989, supra).

In another aspect, the parent is encoded by a polynucleotide thathybridizes under low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 11; or the full-lengthcomplement thereof (Sambrook et al., 1989, supra).

In another aspect, the parent is encoded by a polynucleotide thathybridizes under low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 13; or the full-lengthcomplement thereof (Sambrook et al., 1989, supra).

In another aspect, the parent is encoded by a polynucleotide thathybridizes under low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 15; or the full-lengthcomplement thereof (Sambrook et al., 1989, supra).

In another aspect, the parent is encoded by a polynucleotide thathybridizes under low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 17; or the full-lengthcomplement thereof (Sambrook et al., 1989, supra).

In another aspect, the parent is encoded by a polynucleotide thathybridizes under low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 19; or the full-lengthcomplement thereof (Sambrook et al., 1989, supra).

In another aspect, the parent is encoded by a polynucleotide thathybridizes under low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with the maturepolypeptide coding sequence of SEQ ID NO: 21; or the full-lengthcomplement thereof (Sambrook et al., 1989, supra).

The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, or SEQ ID NO: 21, or a subsequence thereof, aswell as the polypeptide of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, or SEQ ID NO: 22, or a fragment thereof, may be used to designnucleic acid probes to identify and clone DNA encoding a parent fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic DNA or cDNA of a cell of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, e.g., at least 25, atleast 35, or at least 70 nucleotides in length. Preferably, the nucleicacid probe is at least 100 nucleotides in length, e.g., at least 200nucleotides, at least 300 nucleotides, at least 400 nucleotides, atleast 500 nucleotides, at least 600 nucleotides, at least 700nucleotides, at least 800 nucleotides, or at least 900 nucleotides inlength. Both DNA and RNA probes can be used. The probes are typicallylabeled for detecting the corresponding gene (for example, with ³²P, ³H,³⁵S, biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ IDNO: 19, or SEQ ID NO: 21, ora subsequence thereof, the carrier materialis used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ IDNO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQID NO: 19, or SEQ ID NO: 21; (ii) the mature polypeptide coding sequenceof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, or SEQ ID NO: 21; (iii) the full-length complement thereof; or (iv)a subsequence thereof; under very low to very high stringencyconditions. Molecules to which the nucleic acid probe hybridizes underthese conditions can be detected using, for example, X-ray film or anyother detection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, or SEQ ID NO: 21. In another aspect, the nucleic acidprobe is nucleotides 52 to 1673 of SEQ ID NO: 1, nucleotides 52 to 1542of SEQ ID NO: 3, nucleotides 52 to 1542 of SEQ ID NO: 4, nucleotides 79to 1596 of SEQ ID NO: 7, nucleotides 52 to 1371 of SEQ ID NO: 9,nucleotides 55 to 1482 of SEQ ID NO: 11, nucleotides 76 to 1596 of SEQID NO: 13, nucleotides 76 to 1596 of SEQ ID NO: 15, nucleotides 55 to1504 of SEQ ID NO: 17, nucleotides 61 to 1350 of SEQ ID NO: 19, ornucleotides 55 to 1587 of SEQ ID NO: 21. In another aspect, the nucleicacid probe is a polynucleotide that encodes the polypeptide of SEQ IDNO: 2, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22; the maturepolypeptide thereof; or a fragment thereof. In another aspect, thenucleic acid probe is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, or

SEQ ID NO: 21.

In another aspect, the parent is encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, or SEQ ID NO: 4 of at least 60%, e.g., at least65%, at least 70%, at least 75%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100%.

In another aspect, the parent is encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 7 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In another aspect, the parent is encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 9 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In another aspect, the parent is encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 11 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In another aspect, the parent is encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 13 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In another aspect, the parent is encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 15 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In another aspect, the parent is encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 17 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In another aspect, the parent is encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 19 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In another aspect, the parent is encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 21 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In another embodiment, the parent is an allelic variant of the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ IDNO: 22.

The parent may also be a hybrid or chimeric polypeptide in which aregion of parent is replaced with a region of another polypeptide. Inone aspect, the region is a carbohydrate binding domain. Thecarbohydrate binding domain of a parent may be replaced with another(heterologous) carbohydrate binding domain.

The parent may also be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the parent. In one aspect, the other polypeptide is acarbohydrate binding domain. The catalytic domain of a parent without acarbohydrate binding domain may be fused to a carbohydrate bindingdomain. A fusion polypeptide is produced by fusing a polynucleotideencoding another polypeptide to a polynucleotide encoding a parent.Techniques for producing fusion polypeptides are described supra. Afusion polypeptide can further comprise a cleavage site between the twopolypeptides as described supra.

In one embodiment, the parent is a hybrid polypeptide in which thecarbohydrate binding domain of the parent is replaced with a differentcarbohydrate binding domain. In another embodiment, the parent is afusion protein in which a heterologous carbohydrate binding domain isfused to the parent without a carbohydrate binding domain. In oneaspect, the carbohydrate binding domain is fused to the N-terminus ofthe parent. In another aspect, the carbohydrate binding domain is fusedto the C-terminus of the parent. In another aspect, the fusion proteincomprises or consists of SEQ ID NO: 73 or the mature polypeptidethereof. SEQ ID NO: 73 is encoded by SEQ ID NO: 72.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a filamentous fungal cellobiohydrolase. For example,the parent may be a filamentous fungal cellobiohydrolase such as anAspergillus, Chaetomium, Chrysosporium, Myceliophthora, Penicillium,Talaromyces, Thermoascus, or Trichoderma cellobiohydrolase.

In one aspect, the parent is an Aspergillus aculeatus, Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Chaetomium thermophilum, Chrysosporium inops, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium,Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporiumtropicum, Chrysosporium zonatum, Myceliophthora thermophila, Penicilliumemersonii, Penicillium funiculosum, Penicillium purpurogenum,Talaromyces byssochlamydoides, Talaromyces emersonii, Talaromycesleycettanus, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viridecellobiohydrolase.

In another aspect, the parent is a Trichoderma reesei cellobiohydrolase,e.g., the cellobiohydrolase of SEQ ID NO: 2 or the mature polypeptidethereof.

In another aspect, the parent is an Aspergillus fumigatuscellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 8 or themature polypeptide thereof.

In another aspect, the parent is a Thermoascus aurantiacuscellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 10 or themature polypeptide thereof.

In another aspect, the parent is a Penicillium emersonii (Rasamsoniaemersonii) cellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO:12 or the mature polypeptide thereof.

In another aspect, the parent is a Talaromyces leycettanuscellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 14 or themature polypeptide thereof.

In another aspect, the parent is another Talaromyces leycettanuscellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 16 or themature polypeptide thereof.

In another aspect, the parent is a Talaromyces byssochlamydoidescellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 18 or themature polypeptide thereof.

In another aspect, the parent is another Myceliophthora thermophilacellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 20 or themature polypeptide thereof.

In another aspect, the parent is another Chaetomium thermophilumcellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 22 or themature polypeptide thereof.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining acellobiohydrolase variant, comprising: (a) introducing into a parentcellobiohydrolase an alteration at one or more positions correspondingto positions 197, 198, 199, and 200 of the mature polypeptide of SEQ IDNO: 2, wherein the alteration at the one or more positions correspondingto positions 197, 198, and 200 is a substitution and the alteration atthe position corresponding to position 199 is a deletion, and whereinthe variant has cellobiohydrolase activity; and optionally (b)recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent. Any site-directed mutagenesisprocedure can be used in the present invention. There are manycommercial kits available that can be used to prepare variants.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Site-saturation mutagenesis systematically replaces a polypeptide codingsequence with sequences encoding all 19 amino acids at one or more(e.g., several) specific positions (Parikh and Matsumura, 2005, J. Mol.Biol. 352: 621-628).

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga variant of the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide recognized by ahost cell for expression of a polynucleotide encoding a variant of thepresent invention. The promoter contains transcriptional controlsequences that mediate the expression of the variant. The promoter maybe any polynucleotide that shows transcriptional activity in the hostcell including mutant, truncated, and hybrid promoters, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the host cell. Examples of suitablepromoters for directing transcription of the nucleic acid constructs ofthe present invention in a bacterial host cell are the promotersobtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ),Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformispenicillinase gene (penP), Bacillus stearothermophilus maltogenicamylase gene (amyM), Bacillus subtilis levansucrase gene (sacB),Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIAgene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E.coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69:301-315), Streptomyces coelicolor agarase gene (dagA), and prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci.USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983,Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are describedin “Useful proteins from recombinant bacteria” in Gilbert et al., 1980,Scientific American 242: 74-94; and in Sambrook et al., 1989, supra.Examples of tandem promoters are disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thevariant. Any terminator that is functional in the host cell may be usedin the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thevariant. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase,

Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus nigerglucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active variant by catalytic or autocatalytic cleavageof the propeptide from the propolypeptide. The propeptide codingsequence may be obtained from the genes for Bacillus subtilis alkalineprotease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of a variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding the variantwould be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxam ide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris a hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAN/1111permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametesversicolor, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp. 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant,comprising (a) cultivating a recombinant host cell of the presentinvention under conditions conducive for production of the variant; andoptionally (b) recovering the variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cells may be cultivated by shake flask cultivation, or small-scaleor large-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentors in asuitable medium and under conditions allowing the variant to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variants may be detected using methods known in the art that arespecific for the variants. These detection methods include, but are notlimited to, use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. For example, an enzyme assaymay be used to determine the activity of the variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a whole fermentation broth comprising avariant of the present invention is recovered.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a variant of the present invention. Thefermentation broth product further comprises additional ingredients usedin the fermentation process, such as, for example, cells (including, thehost cells containing the gene encoding the variant of the presentinvention which are used to produce the variant), cell debris, biomass,fermentation media and/or fermentation products. In some embodiments,the composition is a cell-killed whole broth containing organic acid(s),killed cells and/or cell debris, and culture medium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compositions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The fermentation broth formulations or cell compositions may furthercomprise multiple enzymatic activities, such as one or more (e.g.,several) enzymes selected from the group consisting of a cellulase, ahemicellulase, a catalase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin. The fermentation broth formulations or cell compositions mayalso comprise one or more (e.g., several) enzymes selected from thegroup consisting of a hydrolase, an isomerase, a ligase, a lyase, anoxidoreductase, or a transferase, e.g., an alpha-galactosidase,alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase,beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase,catalase, cellobiohydrolase, cellulase, chitinase, cutinase,cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase,esterase, glucoamylase, invertase, laccase, lipase, mannosidase,mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)).In some embodiments, the cell-killed whole broth or composition containsthe spent cell culture medium, extracellular enzymes, and killedfilamentous fungal cells. In some embodiments, the microbial cellspresent in the cell-killed whole broth or composition can bepermeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Enzyme Compositions

The present invention also relates to compositions comprising a variantof the present invention. Preferably, the compositions are enriched insuch a variant. The term “enriched” indicates that the cellobiohydrolaseactivity of the composition has been increased, e.g., with an enrichmentfactor of at least 1.1.

The compositions may comprise a variant of the present invention as themajor enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (e.g., several) enzymes selected fromthe group consisting of a cellulase, a hemicellulase, a GH61 polypeptidehaving cellulolytic enhancing activity, a catalase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin. The compositions may also comprise one ormore (e.g., several) enzymes selected from the group consisting of ahydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or atransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase. Thecompositions may be prepared in accordance with methods known in the artand may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Uses

The present invention is also directed to the following processes forusing the variants having cellobiohydrolase I activity of the presentinvention, or compositions thereof.

The present invention also relates to processes for degrading acellulosic material, comprising: treating the cellulosic material withan enzyme composition in the presence of a cellobiohydrolase variant ofthe present invention. In one aspect, the processes further compriserecovering the degraded cellulosic material. Soluble products ofdegradation of the cellulosic material can be separated from insolublecellulosic material using a method known in the art such as, forexample, centrifugation, filtration, or gravity settling.

The present invention also relates to processes of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of acellobiohydrolase variant of the present invention; (b) fermenting thesaccharified cellulosic material with one or more (e.g., several)fermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.

The present invention also relates to processes of fermenting acellulosic material, comprising: fermenting the cellulosic material withone or more (e.g., several) fermenting microorganisms, wherein thecellulosic material is saccharified with an enzyme composition in thepresence of a cellobiohydrolase variant of the present invention. In oneaspect, the fermenting of the cellulosic material produces afermentation product. In another aspect, the processes further compriserecovering the fermentation product from the fermentation.

The processes of the present invention can be used to saccharify thecellulosic material to fermentable sugars and to convert the fermentablesugars to many useful fermentation products, e.g., fuel (ethanol,n-butanol, isobutanol, biodiesel, jet fuel) and/or platform chemicals(e.g., acids, alcohols, ketones, gases, oils, and the like). Theproduction of a desired fermentation product from the cellulosicmaterial typically involves pretreatment, enzymatic hydrolysis(saccharification), and fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using methods conventional in the art.Moreover, the processes of the present invention can be implementedusing any conventional biomass processing apparatus configured tooperate in accordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehanand Himmel, 1999, Biotechnol. Prog. 15: 817-827). HHF involves aseparate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd et al., 2002,Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein thatany method known in the art comprising pretreatment, enzymatichydrolysis (saccharification), fermentation, or a combination thereof,can be used in the practicing the processes of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (deCastilhos Corazza et al., 2003, Acta Scientiarum. Technology 25: 33-38;Gusakov and Sinitsyn, 1985, Enz. Microb. Technol. 7: 346-352), anattrition reactor (Ryu and Lee, 1983, Biotechnol. Bioeng. 25: 53-65).Additional reactor types include fluidized bed, upflow blanket,immobilized, and extruder type reactors for hydrolysis and/orfermentation.

Pretreatment.

In practicing the processes of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents of the cellulosic material (Chandra et al., 2007, Adv.Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Adv.Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009,Bioresource Technol. 100: 10-18; Mosier et al., 2005, BioresourceTechnol. 96: 673-686; Taherzadeh and Karimi, 2008, Int. J. Mol. Sci. 9:1621-1651; Yang and Wyman, 2008, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, sieving, pre-soaking, wetting, washing, and/or conditioningprior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment.

In steam pretreatment, the cellulosic material is heated to disrupt theplant cell wall components, including lignin, hemicellulose, andcellulose to make the cellulose and other fractions, e.g.,hemicellulose, accessible to enzymes. The cellulosic material is passedto or through a reaction vessel where steam is injected to increase thetemperature to the required temperature and pressure and is retainedtherein for the desired reaction time. Steam pretreatment is preferablyperformed at 140-250° C., e.g., 160-200° C. or 170-190° C., where theoptimal temperature range depends on optional addition of a chemicalcatalyst. Residence time for the steam pretreatment is preferably 1-60minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes, or 4-10minutes, where the optimal residence time depends on the temperature andoptional addition of a chemical catalyst. Steam pretreatment allows forrelatively high solids loadings, so that the cellulosic material isgenerally only moist during the pretreatment. The steam pretreatment isoften combined with an explosive discharge of the material after thepretreatment, which is known as steam explosion, that is, rapid flashingto atmospheric pressure and turbulent flow of the material to increasethe accessible surface area by fragmentation (Duff and Murray, 1996,Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl.Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No.2002/0164730). During steam pretreatment, hemicellulose acetyl groupsare cleaved and the resulting acid autocatalyzes partial hydrolysis ofthe hemicellulose to monosaccharides and oligosaccharides. Lignin isremoved to only a limited extent. Chemical Pretreatment: The term“chemical treatment” refers to any chemical pretreatment that promotesthe separation and/or release of cellulose, hemicellulose, and/orlignin. Such a pretreatment can convert crystalline cellulose toamorphous cellulose. Examples of suitable chemical pretreatmentprocesses include, for example, dilute acid pretreatment, limepretreatment, wet oxidation, ammonia fiber/freeze expansion (AFEX),ammonia percolation (APR), ionic liquid, and organosolv pretreatments.

A chemical catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) issometimes added prior to steam pretreatment, which decreases the timeand temperature, increases the recovery, and improves enzymatichydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol.129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol.113-116: 509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39:756-762). In dilute acid pretreatment, the cellulosic material is mixedwith dilute acid, typically H₂SO₄, and water to form a slurry, heated bysteam to the desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technology 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze expansion (AFEX) pretreatment.

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier etal., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatmentmethods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber expansion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technology 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. Biotechnol. 105-108: 69-85, and Mosier etal., 2005, Bioresource Technology 96: 673-686, and U.S. PublishedApplication 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt. % acid, e.g., 0.05 to 5 wt. % acidor 0.1 to 2 wt. % acid. The acid is contacted with the cellulosicmaterial and held at a temperature in the range of preferably 140-200°C., e.g., 165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, e.g., 20-70 wt %or 30-60 wt %, such as around 40 wt %. The pretreated cellulosicmaterial can be unwashed or washed using any method known in the art,e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically)and chemically. Mechanical or physical pretreatment can be coupled withsteaming/steam explosion, hydrothermolysis, dilute or mild acidtreatment, high temperature, high pressure treatment, irradiation (e.g.,microwave irradiation), or combinations thereof. In one aspect, highpressure means pressure in the range of preferably about 100 to about400 psi, e.g., about 150 to about 250 psi. In another aspect, hightemperature means temperature in the range of about 100 to about 300°C., e.g., about 140 to about 200° C. In a preferred aspect, mechanicalor physical pretreatment is performed in a batch-process using a steamgun hydrolyzer system that uses high pressure and high temperature asdefined above, e.g., a Sunds Hydrolyzer available from Sunds DefibratorAB, Sweden. The physical and chemical pretreatments can be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto physical (mechanical) or chemical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Adv. Appl. Microbiol.39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass:a review, in Enzymatic Conversion of Biomass for Fuels Production,Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS SymposiumSeries 566, American Chemical Society, Washington, D.C., chapter 15;Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanolproduction from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996, Enz.Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Adv.Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosicmaterial, e.g., pretreated, is hydrolyzed to break down cellulose and/orhemicellulose to fermentable sugars, such as glucose, cellobiose,xylose, xylulose, arabinose, mannose, galactose, and/or solubleoligosaccharides. The hydrolysis is performed enzymatically by an enzymecomposition in the presence of a cellobiohydrolase variant of thepresent invention. The enzymes of the compositions can be addedsimultaneously or sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzymes(s), i.e., optimalfor the enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic material is fed gradually to,for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 120 hours, e.g., about 16 to about 72 hours or about24 to about 48 hours. The temperature is in the range of preferablyabout 25° C. to about 70° C., e.g., about 30° C. to about 65° C., about40° C. to about 60° C., or about 50° C. to about 55° C. The pH is in therange of preferably about 3 to about 8, e.g., about 3.5 to about 7,about 4 to about 6, or about 4.5 to about 5.5. The dry solids content isin the range of preferably about 5 to about 50 wt %, e.g., about 10 toabout 40 wt % or about 20 to about 30 wt %.

The enzyme compositions can comprise any protein useful in degrading thecellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a ligninolytic enzyme, anoxidoreductase, a pectinase, a protease, and a swollenin. In anotheraspect, the cellulase is preferably one or more (e.g., several) enzymesselected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase. In another aspect, thehemicellulase is preferably one or more (e.g., several) enzymes selectedfrom the group consisting of an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase. In another aspect, the oxidoreductase is preferably one ormore (e.g., several) enzymes selected from the group consisting of acatalase, a laccase, and a peroxidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises aGH61 polypeptide having cellulolytic enhancing activity. In anotheraspect, the enzyme composition comprises an endoglucanase and a GH61polypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises a cellobiohydrolase and a GH61polypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises a beta-glucosidase and a GH61polypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase and acellobiohydrolase. In another aspect, the enzyme composition comprisesan endoglucanase and a cellobiohydrolase I, a cellobiohydrolase II, or acombination of a cellobiohydrolase I and a cellobiohydrolase II. Inanother aspect, the enzyme composition comprises an endoglucanase and abeta-glucosidase. In another aspect, the enzyme composition comprises abeta-glucosidase and a cellobiohydrolase. In another aspect, the enzymecomposition comprises a beta-glucosidase and a cellobiohydrolase I, acellobiohydrolase II, or a combination of a cellobiohydrolase I and acellobiohydrolase II In another aspect, the enzyme composition comprisesan endoglucanase, a GH61 polypeptide having cellulolytic enhancingactivity, and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase, a GH61 polypeptide havingcellulolytic enhancing activity, and a cellobiohydrolase I, acellobiohydrolase II, or a combination of a cellobiohydrolase I and acellobiohydrolase II. In another aspect, the enzyme compositioncomprises an endoglucanase, a beta-glucosidase, and a GH61 polypeptidehaving cellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a beta-glucosidase, a GH61 polypeptide havingcellulolytic enhancing activity, and a cellobiohydrolase. In anotheraspect, the enzyme composition comprises a beta-glucosidase, a GH61polypeptide having cellulolytic enhancing activity, and acellobiohydrolase I, a cellobiohydrolase II, or a combination of acellobiohydrolase I and a cellobiohydrolase II. In another aspect, theenzyme composition comprises an endoglucanase, a beta-glucosidase, and acellobiohydrolase. In another aspect, the enzyme composition comprisesan endoglucanase, a beta-glucosidase, and a cellobiohydrolase I, acellobiohydrolase II, or a combination of a cellobiohydrolase I and acellobiohydrolase II. In another aspect, the enzyme compositioncomprises an endoglucanase, a cellobiohydrolase, a beta-glucosidase, anda GH61 polypeptide having cellulolytic enhancing activity. In anotheraspect, the enzyme composition comprises an endoglucanase, abeta-glucosidase, a GH61 polypeptide having cellulolytic enhancingactivity, and a cellobiohydrolase I, a cellobiohydrolase II, or acombination of a cellobiohydrolase I and a cellobiohydrolase II.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In apreferred aspect, the xylanase is a Family 10 xylanase. In anotherpreferred aspect, the xylanase is a Family 11 xylanase. In anotheraspect, the enzyme composition comprises a xylosidase (e.g.,beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a ligninolytic enzyme. In apreferred aspect, the ligninolytic enzyme is a manganese peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a ligninperoxidase. In another preferred aspect, the ligninolytic enzyme is aH₂O₂-producing enzyme. In another aspect, the enzyme compositioncomprises a pectinase. In another aspect, the enzyme compositioncomprises an oxidoreductase. In another preferred aspect, theoxidoreductase is a catalase. In another preferred aspect, theoxidoreductase is a laccase. In another preferred aspect, theoxidoreductase is a peroxidase. In another aspect, the enzymecomposition comprises a protease. In another aspect, the enzymecomposition comprises a swollenin.

In the processes of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more (e.g., several) components of the enzyme composition may benative proteins, recombinant proteins, or a combination of nativeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. It is understood herein that therecombinant proteins may be heterologous (e.g., foreign) and native tothe host cell. One or more (e.g., several) components of the enzymecomposition may be produced as monocomponents, which are then combinedto form the enzyme composition. The enzyme composition may be acombination of multicomponent and monocomponent protein preparations.

The enzymes used in the processes of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

The optimum amounts of the enzymes and the cellobiohydrolase variantdepend on several factors including, but not limited to, the mixture ofcomponent cellulolytic enzymes and/or hemicellulolytic enzymes, thecellulosic material, the concentration of cellulosic material, thepretreatment(s) of the cellulosic material, temperature, time, pH, andinclusion of fermenting organism (e.g., for SimultaneousSaccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme to the cellulosic material is about 0.5 to about 50 mg, e.g.,about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5to about 10 mg per g of the cellulosic material.

In another aspect, an effective amount of a cellobiohydrolase variant tothe cellulosic material is about 0.01 to about 50.0 mg, e.g., about 0.01to about 40 mg, about 0.01 to about 30 mg, about 0.01 to about 20 mg,about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.025 toabout 1.5 mg, about 0.05 to about 1.25 mg, about 0.075 to about 1.25 mg,about 0.1 to about 1.25 mg, about 0.15 to about 1.25 mg, or about 0.25to about 1.0 mg per g of the cellulosic material.

In another aspect, an effective amount of a cellobiohydrolase variant tocellulolytic or hemicellulolytic enzyme is about 0.005 to about 1.0 g,e.g., about 0.01 to about 1.0 g, about 0.15 to about 0.75 g, about 0.15to about 0.5 g, about 0.1 to about 0.5 g, about 0.1 to about 0.25 g, orabout 0.05 to about 0.2 g per g of cellulolytic or hemicellulolyticenzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material, e.g., GH61 polypeptides havingcellulolytic enhancing activity, (collectively hereinafter “polypeptideshaving enzyme activity”) can be derived or obtained from any suitableorigin, including, archaeal, bacterial, fungal, yeast, plant, or animalorigin. The term “obtained” also means herein that the enzyme may havebeen produced recombinantly in a host organism employing methodsdescribed herein, wherein the recombinantly produced enzyme is eithernative or foreign to the host organism or has a modified amino acidsequence, e.g., having one or more (e.g., several) amino acids that aredeleted, inserted and/or substituted, i.e., a recombinantly producedenzyme that is a mutant and/or a fragment of a native amino acidsequence or an enzyme produced by nucleic acid shuffling processes knownin the art. Encompassed within the meaning of a native enzyme arenatural variants and within the meaning of a foreign enzyme are variantsobtained by, for example, site-directed mutagenesis or shuffling.

A polypeptide having enzyme activity may be a bacterial polypeptide. Forexample, the polypeptide may be a Gram positive bacterial polypeptidesuch as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus,Caldicellulosiruptor, Acidothermus, Thermobifidia, or Oceanobacilluspolypeptide having enzyme activity, or a Gram negative bacterialpolypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma polypeptide having enzyme activity.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide having enzyme activity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In one aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide having enzyme activity.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporiummerdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielaviaspededonium, Thielavia setosa, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaeasaccata polypeptide having enzyme activity.

Chemically modified or protein engineered mutants of polypeptides havingenzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host can be a heterologous host (enzyme is foreign tohost), but the host may under certain conditions also be a homologoushost (enzyme is native to host). Monocomponent cellulolytic proteins mayalso be prepared by purifying such a protein from a fermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes A/S),CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S),CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), SPEZYME™ CP(Genencor Int.), ACCELLERASE™ TRIO (DuPont), FILTRASE® NL (DSM);METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Röhm GmbH), or ALTERNAFUEL®CMAX3™ (Dyadic International, Inc.). The cellulolytic enzyme preparationis added in an amount effective from about 0.001 to about 5.0 wt. % ofsolids, e.g., about 0.025 to about 4.0 wt. % of solids or about 0.005 toabout 2.0 wt. % of solids.

Examples of bacterial endoglucanases that can be used in the processesof the present invention, include, but are not limited to, Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655; WO 00/70031; WO05/093050), Erwinia carotovara endoglucanase (Saarilahti et al., 1990,Gene 90: 9-14), Thermobifida fusca endoglucanase III (WO 05/093050), andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei Cel7B endoglucanase I (GenBank:M15665), Trichoderma reeseiendoglucanase II (Saloheimo et al., 1988, Gene 63:11-22), Trichodermareesei Cel5A endoglucanase II (GenBank:M19373), Trichoderma reeseiendoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64:555-563, GenBank:AB003694), Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228,GenBank:Z33381), Aspergillus aculeatus endoglucanase (Ooi et al., 1990,Nucleic Acids Research 18: 5884), Aspergillus kawachii endoglucanase(Sakamoto et al., 1995, Current Genetics 27: 435-439), Fusariumoxysporum endoglucanase (GenBank:L29381), Humicola grisea var.thermoidea endoglucanase (GenBank:AB003107), Melanocarpus albomycesendoglucanase (GenBank:MAL515703), Neurospora crassa endoglucanase(GenBank:XM_324477), Humicola insolens endoglucanase V, Myceliophthorathermophila CBS 117.65 endoglucanase, Thermoascus aurantiacusendoglucanase I (GenBank:AF487830), Trichoderma reesei strain No.VTT-D-80133 endoglucanase (GenBank:M15665), and Penicillium pinophilumendoglucanase (WO 2012/062220).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Aspergillus fumigatus cellobiohydrolase I (WO2013/028928), Aspergillus fumigatus cellobiohydrolase II (WO2013/028928), Chaetomium thermophilum cellobiohydrolase I, Chaetomiumthermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolaseI, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Penicillium occitanis cellobiohydrolase I (GenBank:AY690482),Talaromyces emersonii cellobiohydrolase I (GenBank:AF439936), Thielaviahyrcanie cellobiohydrolase II (WO 2010/141325), Thielavia terrestriscellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 02/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO2011/035029), and Trichophaea saccata (WO 2007/019442).

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is an Aspergillus oryzae beta-glucosidase variant BGfusion protein (WO 2008/057637) or an Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637).

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat, 1991, Biochem. J. 280: 309-316,and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO2008/008070, WO 2008/008793, U.S. Pat. Nos. 5,457,046, 5,648,263, and5,686,593.

In the processes of the present invention, any GH61 polypeptide havingcellulolytic enhancing activity can be used as a component of the enzymecomposition.

Examples of GH61 polypeptides useful in the processes of the presentinvention include, but are not limited to, GH61 polypeptides fromThielavia terrestris (WO 2005/074647, WO 2008/148131, and WO2011/035027), Thermoascus aurantiacus (WO 2005/074656 and WO2010/065830), Trichoderma reesei (WO 2007/089290 and WO 2012/149344),Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO2009/085864, WO 2009/085868, and WO 2009/033071), Aspergillus fumigatus(WO 2010/138754), Penicillium pinophilum (WO 2011/005867), Thermoascussp. (WO 2011/039319), Penicillium sp. (emersonii) (WO 2011/041397 and WO2012/000892), Thermoascus crustaceous (WO 2011/041504), Aspergillusaculeatus (WO 2012/125925), Thermomyces lanuginosus (WO 2012/113340, WO2012/129699, WO 2012/130964, and WO 2012/129699), Aurantiporusalborubescens (WO 2012/122477), Trichophaea saccata (WO 2012/122477),Penicillium thomii (WO 2012/122477), Talaromyces stipitatus (WO2012/135659), Humicola insolens (WO 2012/146171), Malbranchea cinnamomea(WO 2012/101206), Talaromyces leycettanus (WO 2012/101206), andChaetomium thermophilum (WO 2012/101206), and Talaromyces thermophilus(WO 2012/129697 and WO 2012/130950).

In one aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a soluble activating divalent metalcation according to WO 2008/151043, e.g., manganese or copper.

In another aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a dioxy compound, a bicyliccompound, a heterocyclic compound, a nitrogen-containing compound, aquinone compound, a sulfur-containing compound, or a liquor obtainedfrom a pretreated cellulosic material such as pretreated corn stover (WO2012/021394, WO 2012/021395, WO 2012/021396, WO 2012/021399, WO2012/021400, WO 2012/021401, WO 2012/021408, and WO 2012/021410).

The dioxy compound may include any suitable compound containing two ormore oxygen atoms. In some aspects, the dioxy compounds contain asubstituted aryl moiety as described herein. The dioxy compounds maycomprise one or more (e.g., several) hydroxyl and/or hydroxylderivatives, but also include substituted aryl moieties lacking hydroxyland hydroxyl derivatives. Non-limiting examples of the dioxy compoundsinclude pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoicacid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid;methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone;2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid;4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethylgallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1,4-diol;(croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol;3-ethyoxy-1,2-propanediol; 2,4,4′-trihydroxybenzophenone;cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione;dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate;4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or asalt or solvate thereof.

The bicyclic compound may include any suitable substituted fused ringsystem as described herein. The compounds may comprise one or more(e.g., several) additional rings, and are not limited to a specificnumber of rings unless otherwise stated. In one aspect, the bicycliccompound is a flavonoid. In another aspect, the bicyclic compound is anoptionally substituted isoflavonoid. In another aspect, the bicycliccompound is an optionally substituted flavylium ion, such as anoptionally substituted anthocyanidin or optionally substitutedanthocyanin, or derivative thereof. Non-limiting examples of thebicyclic compounds include epicatechin; quercetin; myricetin; taxifolin;kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin;cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

The heterocyclic compound may be any suitable compound, such as anoptionally substituted aromatic or non-aromatic ring comprising aheteroatom, as described herein. In one aspect, the heterocyclic is acompound comprising an optionally substituted heterocycloalkyl moiety oran optionally substituted heteroaryl moiety. In another aspect, theoptionally substituted heterocycloalkyl moiety or optionally substitutedheteroaryl moiety is an optionally substituted 5-memberedheterocycloalkyl or an optionally substituted 5-membered heteroarylmoiety. In another aspect, the optionally substituted heterocycloalkylor optionally substituted heteroaryl moiety is an optionally substitutedmoiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl,oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl,dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl,pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl,piperidinyl, and oxepinyl. In another aspect, the optionally substitutedheterocycloalkyl moiety or optionally substituted heteroaryl moiety isan optionally substituted furanyl. Non-limiting examples of theheterocyclic compounds include(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one;4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone;[1,2-dihydroxyethyl]furan-2,3,4(5H)-trione; α-hydroxy-γ-butyrolactone;ribonic γ-lactone; aldohexuronicaldohexuronic acid γ-lactone; gluconicacid δ-lactone; 4-hydroxycoumarin; dihydrobenzofuran;5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone;5,6-dihydro-2H-pyran-2-one; and5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvatethereof.

The nitrogen-containing compound may be any suitable compound with oneor more nitrogen atoms. In one aspect, the nitrogen-containing compoundcomprises an amine, imine, hydroxylamine, or nitroxide moiety.Non-limiting examples of the nitrogen-containing compounds includeacetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol;1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy;5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; andmaleamic acid; or a salt or solvate thereof.

The quinone compound may be any suitable compound comprising a quinonemoiety as described herein. Non-limiting examples of the quinonecompounds include 1,4-benzoquinone; 1,4-naphthoquinone;2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4-benzoquinone orcoenzyme Q₀; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone;1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione oradrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; pyrroloquinolinequinone; or a salt or solvate thereof.

The sulfur-containing compound may be any suitable compound comprisingone or more sulfur atoms. In one aspect, the sulfur-containing comprisesa moiety selected from thionyl, thioether, sulfinyl, sulfonyl,sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limitingexamples of the sulfur-containing compounds include ethanethiol;2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonic acid;benzenethiol; benzene-1,2-dithiol; cysteine; methionine; glutathione;cystine; or a salt or solvate thereof.

In one aspect, an effective amount of such a compound described above isadded to cellulosic material at a molar ratio of the compound toglucosyl units of cellulose of about 10⁻⁶ to about 10, e.g., about 10⁻⁶to about 7.5, about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5, about 10⁻⁶to about 1, about 10⁻⁵ to about 1, about 10⁻⁵ to about 10⁻¹, about 10⁻⁴to about 10⁻¹, about 10⁻³ to about 10⁻¹, or about 10⁻³ to about 10⁻². Inanother aspect, an effective amount of such a compound is about 0.1 μMto about 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μM to about0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μMto about 50 mM, about 10 μM to about 25 mM, about 50 μM to about 25 mM,about 10 μM to about 10 mM, about 5 μM to about 5 mM, or about 0.1 mM toabout 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described in WO2012/021401, and the soluble contents thereof. A liquor for cellulolyticenhancement of a GH61 polypeptide can be produced by treating alignocellulose or hemicellulose material (or feedstock) by applying heatand/or pressure, optionally in the presence of a catalyst, e.g., acid,optionally in the presence of an organic solvent, and optionally incombination with physical disruption of the material, and thenseparating the solution from the residual solids. Such conditionsdetermine the degree of cellulolytic enhancement obtainable through thecombination of liquor and a GH61 polypeptide during hydrolysis of acellulosic substrate by a cellulolytic enzyme preparation. The liquorcan be separated from the treated material using a method standard inthe art, such as filtration, sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5 g, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about1 g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² gper g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC®HTec3 (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (NovozymesA/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor),ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A(AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit,Wales, UK), DEPOL™ 740 L. (Biocatalysts Limit, Wales, UK), and DEPOL™762P (Biocatalysts Limit, Wales, UK), ALTERNA FUEL 100P (Dyadic), andALTERNA FUEL 200P (Dyadic).

Examples of xylanases useful in the processes of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Thermomyces lanuginosus (GeneSeqP:BAA22485),Talaromyces thermophilus (GeneSeqP:BAA22834), Thielavia terrestris NRRL8126 (WO 2009/079210), and Trichophaea saccata (WO 2011/057083).

Examples of beta-xylosidases useful in the processes of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (SwissProt:Q7SOW4), Trichoderma reesei (UniProtKB/TrEMBL: Q92458), Talaromyces emersonii (SwissProt:Q8X212), and Talaromycesthermophilus (GeneSeqP:BAA22816).

Examples of acetylxylan esterases useful in the processes of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum (UniProt:Q2GWX4), Chaetomium gracile (GeneSeqP:AAB82124), Humicola insolens DSM1800 (WO 2009/073709), Hypocrea jecorina (WO 2005/001036), Myceliophterathermophila (WO 2010/014880), Neurospora crassa (UniProt:q7s259),Phaeosphaeria nodorum (UniProt:QOUHJ1), and Thielavia terrestris NRRL8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in theprocesses of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt:A1D9T4), Neurospora crassa(UniProt:Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729), andThielavia terrestris (WO 2010/053838 and WO 2010/065448).

Examples of arabinofuranosidases useful in the processes of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger (GeneSeqP:AAR94170), Humicola insolens DSM 1800 (WO2006/114094 and WO 2009/073383), and M. giganteus (WO 2006/114094).

Examples of alpha-glucuronidases useful in the processes of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt:alcc12), Aspergillus fumigatus(SwissProt:Q4WW45), Aspergillus niger (UniProt: Q96WX9), Aspergillusterreus (Swiss Prot: QOCJ P9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt:Q8X211), and Trichoderma reesei (UniProt:Q99024).

Examples of oxidoreductases useful in the processes of the presentinvention include, but are not limited to, Aspergillus fumigatuscatalase, Aspergillus lentilus catalase, Aspergillus niger catalase,Aspergillus oryzae catalase, Humicola insolens catalase, Neurosporacrassa catalase, Penicillium emersonii catalase, Scytalidiumthermophilum catalase, Talaromyces stipitatus catalase, Thermoascusaurantiacus catalase, Coprinus cinereus laccase, Myceliophthorathermophila laccase, Polyporus pinsitus laccase, Pycnoporus cinnabarinuslaccase, Rhizoctonia solani laccase, Streptomyces coelicolor laccase,Coprinus cinereus peroxidase, Soy peroxidase, and Royal palm peroxidase.

The polypeptides having enzyme activity used in the processes of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, C A, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, N Y, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Fermentation.

The fermentable sugars obtained from the hydrolyzed cellulosic materialcan be fermented by one or more (e.g., several) fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on economics, i.e., costs per equivalent sugarpotential, and recalcitrance to enzymatic conversion.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Yeast includestrains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candidasonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Xylose fermenting yeast include strains of Candida, preferably C.sheatae or C. sonorensis; and strains of Pichia, e.g., P. stipitis, suchas P. stipitis CBS 5773. Pentose fermenting yeast include strains ofPachysolen, preferably P. tannophilus. Organisms not capable offermenting pentose sugars, such as xylose and arabinose, may begenetically modified to do so by methods known in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, G. P., 1996, Cellulose bioconversion technology,in Handbook on Bioethanol: Production and Utilization, Wyman, C. E.,ed., Taylor & Francis, Washington, D.C., 179-212).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In an aspect, the fermenting microorganism has been genetically modifiedto provide the ability to ferment pentose sugars, such as xyloseutilizing, arabinose utilizing, and xylose and arabinose co-utilizingmicroorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Appl.Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Appl. Environ.Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993, Appl. Microbiol.Biotechnol. 38: 776-783; Walfridsson et al., 1995, Appl. Environ.Microbiol. 61: 4184-4190; Kuyper et al., 2004, FEMS Yeast Research 4:655-664; Beall et al., 1991, Biotech. Bioeng. 38: 296-303; Ingram etal., 1998, Biotechnol. Bioeng. 58: 204-214; Zhang et al., 1995, Science267: 240-243; Deanda et al., 1996, Appl. Environ. Microbiol. 62:4465-4470; WO 03/062430).

In one aspect, the fermenting organism comprises a polynucleotideencoding a variant of the present invention.

In another aspect, the fermenting organism comprises one or morepolynucleotides encoding one or more cellulolytic enzymes,hemicellulolytic enzymes, and accessory enzymes described herein.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol,methanol, ethylene glycol, 1,3-propanediol [propylene glycol],butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane), acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); anamino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide(CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); anorganic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbicacid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaricacid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); and polyketide.

In one aspect, the fermentation product is an alcohol. The term“alcohol” encompasses a substance that contains one or more hydroxylmoieties. The alcohol can be, but is not limited to, n-butanol,isobutanol, ethanol, methanol, arabinitol, butanediol, ethylene glycol,glycerin, glycerol, 1,3-propanediol, sorbitol, xylitol. See, forexample, Gong et al., 1999, Ethanol production from renewable resources,in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira andJonas, 2002, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam and Singh,1995, Process Biochemistry 30(2): 117-124; Ezeji et al., 2003, WorldJournal of Microbiology and Biotechnology 19(6): 595-603.

In another aspect, the fermentation product is an alkane. The alkane maybe an unbranched or a branched alkane. The alkane can be, but is notlimited to, pentane, hexane, heptane, octane, nonane, decane, undecane,or dodecane.

In another aspect, the fermentation product is a cycloalkane. Thecycloalkane can be, but is not limited to, cyclopentane, cyclohexane,cycloheptane, or cyclooctane.

In another aspect, the fermentation product is an alkene. The alkene maybe an unbranched or a branched alkene. The alkene can be, but is notlimited to, pentene, hexene, heptene, or octene.

In another aspect, the fermentation product is an amino acid. Theorganic acid can be, but is not limited to, aspartic acid, glutamicacid, glycine, lysine, serine, or threonine. See, for example, Richardand Margaritis, 2004, Biotechnology and Bioengineering 87(4): 501-515.

In another aspect, the fermentation product is a gas. The gas can be,but is not limited to, methane, H₂, CO₂, or CO. See, for example,Kataoka et al., 1997, Water Science and Technology 36(6-7): 41-47; andGunaseelan, 1997, Biomass and Bioenergy 13(1-2): 83-114.

In another aspect, the fermentation product is isoprene.

In another aspect, the fermentation product is a ketone. The term“ketone” encompasses a substance that contains one or more ketonemoieties. The ketone can be, but is not limited to, acetone.

In another aspect, the fermentation product is an organic acid. Theorganic acid can be, but is not limited to, acetic acid, acetonic acid,adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid,formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronicacid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lacticacid, malic acid, malonic acid, oxalic acid, propionic acid, succinicacid, or xylonic acid. See, for example, Chen and Lee, 1997, Appl.Biochem. Biotechnol. 63-65: 435-448.

In another aspect, the fermentation product is polyketide.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce acellobiohydrolase variant in recoverable quantities. The variant may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the variant may be used as such for improving thequality of a food or feed, e.g., improving nutritional value,palatability, and rheological properties, or to destroy an antinutritivefactor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a variant may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or moreexpression constructs encoding a variant into the plant host genome orchloroplast genome and propagating the resulting modified plant or plantcell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a variant operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the variant is desired tobe expressed (Sticklen, 2008, Nature Reviews 9: 433-443). For instance,the expression of the gene encoding a variant may be constitutive orinducible, or may be developmental, stage or tissue specific, and thegene product may be targeted to a specific tissue or plant part such asseeds or leaves. Regulatory sequences are, for example, described byTague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a variant in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a variant. For instance, Xu et al.,1993, supra, disclose the use of the first intron of the rice actin 1gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a variant can be introducedinto a particular plant variety by crossing, without the need for everdirectly transforming a plant of that given variety. Therefore, thepresent invention encompasses not only a plant directly regenerated fromcells which have been transformed in accordance with the presentinvention, but also the progeny of such plants. As used herein, progenymay refer to the offspring of any generation of a parent plant preparedin accordance with the present invention. Such progeny may include a DNAconstruct prepared in accordance with the present invention. Crossingresults in the introduction of a transgene into a plant line by crosspollinating a starting line with a donor plant line. Non-limitingexamples of such steps are described in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a variant ofthe present invention comprising: (a) cultivating a transgenic plant ora plant cell comprising a polynucleotide encoding the variant underconditions conducive for production of the variant; and optionally (b)recovering the variant.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Strains

Aspergillus oryzae strain MT3568 was used as a host for expression ofthe Trichoderma reesei gene encoding cellobiohydrolase I and a variantthereof. A. oryzae MT3568 is an amdS (acetamidase) disrupted genederivative of Aspergillus oryzae JaL355 (WO 2002/40694) in which pyrGauxotrophy was restored by disrupting the A. oryzae acetamidase (amdS)gene.

Media and Solutions

COVE sucrose plates or slants were composed of 342 g of sucrose, 20 g ofagar powder, 20 ml of COVE salt solution, and deionized water to 1liter. The medium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 60° C. and then acetamide to 10 mM, CsCl to 15 mM,and TRITON® X-100 (50 μl/500 ml) were added.

COVE salt solution was composed of 26 g of MgSO₄.7H₂O, 26 g of KCl, 26 gof KH₂PO₄, 50 ml of COVE trace metals solution, and deionized water to 1liter. COVE trace metals solution was composed of 0.04 g ofNa₂B₄O₇.10H₂O, 0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g of Na₂MoO₄.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1liter.

DAP-4C medium was composed of 20 g of dextrose, 10 g of maltose, 11 g ofMgSO₄.7H₂O, 1 g of KH₂PO₄, 2 g of citric acid, 5.2 g of K₃PO₄. H₂O, 0.5g of yeast extract (Difco), 1 ml of antifoam, 0.5 ml of KU6 trace metalssolution, 2.5 g of CaCO₃, and deionized water to 1 liter. The medium wassterilized by autoclaving at 15 psi for 15 minutes (BacteriologicalAnalytical Manual, 8th Edition, Revision A, 1998). Before use, 3.5 ml ofsterile 50% (NH₄)₂HPO₄ and 5 ml of sterile 20% lactic acid were addedper 150 ml.

G2-Gly medium was composed of 18 g of yeast extract, 24 g of glycerol(86-88%), 1 ml of antifoam, and deionized water to 1 liter.

KU6 trace metals solution was composed of 0.13 g of NiCl₂, 2.5 g ofCuSO₄.5H₂O, 13.9 g of FeSO₄.7H₂O, 8.45 g of MnSO₄.H₂O, 6.8 g of ZnCl₂, 3g of citric acid, and deionized water to 1 liter.

LB medium was composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,10 g of sodium chloride, and deionized water to 1 liter.

LB plates were composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,10 g of sodium chloride, 15 g of Bacto-agar, and deionized water to 1liter.

PDA plates were composed of potato infusion made by boiling 300 g ofsliced (washed but unpeeled) potatoes in water for 30 minutes and thendecanting or straining the broth through cheesecloth. Distilled waterwas then added until the total volume of the suspension was 1 liter.Then 20 g of dextrose and 20 g of agar powder were added. The medium wassterilized by autoclaving at 15 psi for 15 minutes (BacteriologicalAnalytical Manual, 8th Edition, Revision A, 1998).

TAE buffer was composed of 40 mM Tris base, 20 mM sodium acetate, and 1mM disodium EDTA.

YP+2% glucose medium was composed of 1% yeast extract, 2% peptone, and2% glucose in deionized water.

YP+2% maltose medium was composed of 10 g of yeast extract, 20 g ofpeptone, 20 g of maltose, and deionized water to 1 liter.

Example 1: Source of DNA Sequence Information for Trichoderma reeseiCellobiohydrolase I

The genomic DNA sequence and deduced amino acid sequence of theTrichoderma reesei GH7 cellobiohydrolase I gene is shown in SEQ ID NO: 1and SEQ ID NO: 2, respectively. Genomic sequence information wasgenerated by the U.S. Department of Energy Joint Genome Institute (JGI)and published by Martinez et al., 2008, Nature Biotechnology 26 (5):553-560. The amino acid sequence of the full-length cellobiohydrolase Iis publicly available from the National Center for BiotechnologyInformation (NCBI) and annotated as GenBank: EGR44817.1 (SEQ ID NO: 2).The cDNA sequence and deduced amino acid sequence of the Trichodermareesei cellobiohydrolase I gene is shown in SEQ ID NO: 3 and SEQ ID NO:2, respectively.

Based on the publicly available amino acid sequence, a codon-optimizedsynthetic gene encoding the full-length cellobiohydrolase I wasgenerated for Aspergillus oryzae expression based on the algorithmdeveloped by Gustafsson et al., 2004, Trends in Biotechnology 22 (7):346-353. The codon-optimized coding sequence (SEQ ID NO: 4) wassynthesized by the GENEART® Gene Synthesis service (Life TechnologiesCorp., San Diego. Calif., USA) with a 5′ Bam HI restriction site, a 3′Hind III restriction site, and a Kozac consensus sequence (CACC)situated between the start codon and the Bam HI restriction site.

Example 2: Site-Directed Mutagenesis of the Trichoderma reeseiCellobiohydrolase I

The codon-optimized synthetic gene encoding the T. reeseicellobiohydrolase I was provided in a non-specified kanamycin-resistantE. coli cloning vector. To generate the T. reesei cellobiohydrolase I M6variant (SEQ ID NO: 5 for the mutant DNA sequence and SEQ ID NO: 6 forthe variant), an AAC codon (N197) was replaced with a GCC codon (197A)and an AAC codon (N200) was replaced with a GCC codon (200A). Twosynthetic primers for site-directed mutagenesis were designed as shownbelow using the QUIKCHANGE® Primer Design (Agilent Technologies, Inc.,Wilmington, Del., USA) online tool to introduce the site-directedmutations changing an AAC codon (N197) to a GCC codon (197A) and an AACcodon (N200) to a GCC codon (200A).

Site-directed mutagenesis of the synthetic gene encoding the wild-typeT. reesei cellobiohydrolase was facilitated by two PCR amplifications ofthe kanamycin-resistant E. coli cloning vector provided by GENEART® GeneSynthesis using the primers and procedure described below:

Primer F-N197A: (SEQ ID NO: 23) 5′-GGGAACCCTCGTCGGCCAACGCCAACACCG-3′Primer R-N197A: (SEQ ID NO: 24) 5′-CGGTGTTGGCGTTGGCCGACGAGGGTTCCC-3′Primer F-N200A: (SEQ ID NO: 25) 5′-TCGTCGGCCAACGCCGCCACCGGCATTGGAGG-3′Primer R-N200A: (SEQ ID NO: 26) 5′-CCTCCAATGCCGGTGGCGGCGTTGGCCGACGA-3′

The two mutations were introduced consecutively by PCR using a PHUSION®High-Fidelity PCR Kit (Finnzymes Oy, Espoo, Finland). The PCR solutionwas composed of 10 μl of 5×HF buffer (Finnzymes Oy, Espoo, Finland), 1μl of dNTPs (10 mM), 0.5 μl of PHUSION® DNA polymerase (0.2 units/μl)(Finnzymes Oy, Espoo, Finland), 2.5 μl of primer F-N197A (10 μM), 2.5 μlof primer R-N197A (10 μM), 1 μl of template DNA (GENEART® vector, 10ng/μl), and 32.5 μl of deionized water in a total volume of 50 μl. ThePCR was performed using a PTC-200 DNA Engine (MJ Research Inc., Waltham,Mass., USA) programmed for 1 cycle at 98° C. for 30 seconds; and 16cycles each at 98° C. for 30 seconds, 55° C. for 1 minute, and 72° C.for 4 minutes. The PCR solution was then held at 15° C. until removedfrom the PCR machine.

Following PCR, 10 units of Dpn I were added directly to the PCR solutionand incubated at 37° C. for 1 hour. Then 1 μl of the Dpn I treated PCRsolution was transformed into ONE SHOT® TOP10F′ Chemically Competent E.coli cells (Invitrogen, Carlsbad, Calif., USA) according to themanufacturer's protocol and spread onto LB plates supplemented with 0.05mg of kanamycin per ml. After incubation at 37° C. overnight,transformants were observed growing under selection on the LB kanamycinplates. Two transformants were cultivated in LB medium supplemented with0.05 mg of kanamycin per ml and plasmids were isolated using a QIAPREP®Spin Miniprep Kit (QIAGEN Inc., Valencia, Calif., USA).

The isolated plasmids were sequenced using an Applied Biosystems 3730xlDNA Analyzer (Applied Biosystems, Foster City, Calif., USA) with vectorprimers and a T. reesei cellobiohydrolase I gene specific primer(R-Central), shown below, in order to determine a representative plasmidthat was free of PCR errors and contained the AAC to GCC mutation.

Primer F-vector: (SEQ ID NO: 27) 5′-CGTTGTAAAACGACGGCC-3′Primer R-vector: (SEQ ID NO: 28) 5′-TGTTAATGCAGCTGGCAC-3′Primer R-Central: (SEQ ID NO: 29) 5′-CTTGTCGGAGAACGACGA-3′

One plasmid clone free of PCR errors and containing the AAC (N197) toGCC (197A) mutation was chosen and designated plasmid pN197A.

A second round of PCR was performed to introduce the N200A mutation byPCR using a PHUSION® High-Fidelity PCR Kit. The PCR solution wascomposed of 10 μl of 5×HF buffer, 1 μl of dNTPs (10 mM), 0.5 μl ofPHUSION® DNA polymerase (0.2 units/μl), 2.5 μl of primer F-N200A (10μM), 2.5 μl of primer R-N200A (10 μM), 1 μl of template DNA (pN197A, 10ng/μl), and 32.5 μl of deionized water in a total volume of 50 μl. ThePCR was performed using a PTC-200 DNA Engine programmed for 1 cycle at98° C. for 30 seconds; and 16 cycles each at 98° C. for 30 seconds, 55°C. for 1 minute, and 72° C. for 4 minutes. The PCR solution was thenheld at 15° C. until removed from the PCR machine.

Following PCR, 10 units of Dpn I were added directly to the PCR solutionand incubated at 37° C. for 1 hour. Then 1 μl of the Dpn I treated PCRsolution was transformed into ONE SHOT® TOP10F′ Chemically Competent E.coli cells according to the manufacturer's protocol and spread onto LBplates supplemented with 0.05 mg of kanamycin per ml. After incubationat 37° C. overnight, transformants were observed growing under selectionon the LB kanamycin plates. Two transformants were cultivated in LBmedium supplemented with 0.05 mg of kanamycin per ml and plasmids wereisolated using a QIAPREP® Spin Miniprep Kit.

The isolated plasmids were sequenced using an Applied Biosystems 3730xlDNA Analyzer with primers F-vector, R-vector, and R-Central shown abovein order to determine a representative plasmid that was free of PCRerrors and contained the AAC to GCC mutation.

One plasmid clone free of PCR errors and containing the AAC (N197) toGCC (197A) and ACC (N200) to GCC (200A) mutations was chosen anddesignated plasmid pM6. The variant is designated herein as “M6variant”.

Example 3: Construction of an Aspergillus oryzae Expression VectorContaining a Trichoderma reesei cDNA Sequence Encoding CellobiohydrolaseI

The kanamycin-resistant E. coli cloning vector provided by GENEART® GeneSynthesis encoding the T. reesei cellobiohydrolase I (SEQ ID NO: 4) wasdigested with Fast Digest Bam HI and Hind III (Fermentas Inc., GlenBurnie, Md., USA) according to manufacturer's instructions. The reactionproducts were isolated by 1.0% agarose gel electrophoresis using TAEbuffer where a 1552 bp product band was excised from the gel andpurified using an ILLUSTRA™ GFX™ DNA Purification Kit (GE HealthcareLife Sciences, Brondby, Denmark).

The 1552 bp fragment was then cloned into pDau109 (WO 2005/042735)digested with Bam HI and Hind III using T4 DNA ligase (New EnglandBiolabs, Ipswich, Mass., USA). The Bam HI-Hind III digested pDau109 andthe Bam HI/Hind III fragment containing the T. reesei cellobiohydrolaseI coding sequence were mixed in a molar ratio of 1:3 (i.e., mass ratioapproximately 2.5:1 or 20 ng:50 ng) and ligated with 50 units of T4 DNAligase in 1×T4 DNA ligase buffer (New England Biolabs, Ipswich, Mass.,USA) with 1 mM ATP at 16° C. over-night in accordance with themanufacturer's instructions. Cloning of the T. reesei cellobiohydrolaseI gene into the Bam HI-Hind III digested pDau109 resulted intranscription of the T. reesei cellobiohydrolase I gene under thecontrol of a NA2-tpi double promoter. The NA2-tpi promoter is a modifiedpromoter from the gene encoding the Aspergillus niger neutralalpha-amylase in which the untranslated leader has been replaced by anuntranslated leader from the gene encoding the Aspergillus nidulanstriose phosphate isomerase.

The ligation mixture was transformed into ONE SHOT® TOP10F′ ChemicallyCompetent E. coli cells according to the manufacturer's protocol andspread onto LB plates supplemented with 0.1 mg of ampicillin per ml.After incubation at 37° C. overnight, colonies were observed growingunder selection on the LB ampicillin plates

Insertion of the T. reesei cellobiohydrolase I gene into pDau109 wasverified by PCR on colonies as described below using the followingprimers.

Primer F-pDau109 (SEQ ID NO: 30) 5′-CCCTTGTCGATGCGATGTATC-3′Primer R-pDau109 (SEQ ID NO: 31) 5′-ATCCTCAATTCCGTCGGTCGA-3′

A 1.1× REDDYMIX® Master Mix (Thermo Fisher Scientific, Roskilde,Denmark) was used for the PCR. The PCR solution was composed of 10 μl of1.1× REDDYMIX® Master Mix, 0.5 μl of primer F-pDau109 (10 μM), and 0.5μl of primer R-pDau109 (10 μM). A toothpick was used to transfer a smallamount of cells to the PCR solution. The PCR was performed using aPTC-200 DNA Engine programmed for 1 cycle at 94° C. for 3 minutes; 30cycles each at 94° C. for 30 seconds, 50° C. for 1 minute, and 72° C.for 2 minutes; and 1 cycle at 72° C. for 1 minute. The PCR solution wasthen held at 15° C. until removed from the PCR machine.

The PCR products were analyzed by 1.0% agarose gel electrophoresis usingTAE buffer where a 1860 bp PCR product band was observed confirminginsertion of the T. reesei cellobiohydrolase I coding sequence intopDau109.

An E. coli transformant containing the T. reesei cellobiohydrolase Iplasmid construct was cultivated in LB medium supplemented with 0.1 mgof ampicillin per ml and plasmid DNA was isolated using a QIAPREP® SpinMiniprep Kit. The plasmid was designated pKHJN0036.

Example 4: Construction of an Aspergillus oryzae Expression VectorContaining Trichoderma reesei cDNA Sequence Encoding theCellobiohydrolase I M6 Variant

Plasmid pM6 encoding the T. reesei cellobiohydrolase I M6 variant wasdigested with Fast Digest Bam HI and Hind III according tomanufacturer's instructions. The reaction products were isolated by 1.0%agarose gel electrophoresis using TAE buffer where a 1552 bp productband was excised from the gel and purified using an ILLUSTRA™ GFX™ DNAPurification Kit.

The 1552 bp fragment was then cloned into pDau109 digested with Bam HIand Hind III using T4 DNA ligase. The Bam HI-Hind III digested pDau109and the Bam HI/Hind III fragment containing the T. reeseicellobiohydrolase I M6 variant coding sequence were mixed in a molarratio of 1:3 (i.e., mass ratio approximately 2.5:1 or 20 ng:50 ng) andligated with 50 units of T4 DNA ligase in 1×T4 DNA ligase buffer with 1mM ATP at 16° C. overnight. Cloning of the T. reesei cellobiohydrolase IM6 variant gene into the Bam HI-Hind III digested pDau109 resulted intranscription of the T. reesei cellobiohydrolase I M6 variant gene underthe control of a NA2-tpi double promoter described above.

The ligation mixture was transformed into ONE SHOT® TOP10F′ ChemicallyCompetent E. coli cells according to the manufacturer's protocol andspread onto LB plates supplemented with 0.1 mg of ampicillin per ml.After incubation at 37° C. overnight, colonies were observed growingunder selection on the LB ampicillin plates

Insertion of the T. reesei cellobiohydrolase I M6 variant gene intopDau109 was verified by PCR on colonies as described below using primersF-pDau109 and R-pDau109 (Example 3) shown below.

Primer F-pDau109 (SEQ ID NO: 30) 5′-CCCTTGTCGATGCGATGTATC-3′Primer R-pDau109 (SEQ ID NO: 31) 5′-ATCCTCAATTCCGTCGGTCGA-3′

A 1.1× REDDYMIX® Master Mix was used for the PCR. The PCR solution wascomposed of 10 μl of 1.1× REDDYMIX® Master Mix, 0.5 μl of primerF-pDau109 (10 μM), and 0.5 μl of primer R-pDau109 (10 μM). A toothpickwas used to transfer a small amount of cells to the PCR solution. ThePCR was performed using a PTC-200 DNA Engine programmed for 1 cycle at94° C. for 3 minutes; 30 cycles each at 94° C. for 30 seconds, 50° C.for 1 minute, and 72° C. for 2 minutes; and 1 cycle at 72° C. for 1minute. The PCR solution was then held at 15° C. until removed from thePCR machine.

The PCR reaction products were analyzed by 1.0% agarose gelelectrophoresis using TAE buffer where a 1860 bp PCR product band wasobserved confirming insertion of the T. reesei cellobiohydrolase I M6variant coding sequence into pDau109.

An E. coli transformant containing the T. reesei cellobiohydrolase I M6variant plasmid construct was cultivated in LB medium supplemented with0.1 mg of ampicillin per ml and plasmid was isolated using a QIAPREP®Spin Miniprep Kit. The plasmid was designated pKHJN0059.

Example 5: Expression of the Wild-Type Trichoderma reeseiCellobiohydrolase I

The expression plasmid pKHJN0036 was transformed into Aspergillus oryzaeMT3568 protoplasts according to Christensen et al., 1988, Biotechnology6, 1419-1422 and WO 2004/032648. A. oryzae MT3568 protoplasts wereprepared according to the method of EP 0238023 B1, pages 14-15.

Transformants were purified on COVE sucrose plates through singleconidia prior to sporulating them on PDA plates. Spores of thetransformants were inoculated into 96 deep well plates containing 0.75ml of YP+2% glucose medium and incubated stationary at 30° C. for 4days. Production of the T. reesei cellobiohydrolase I by thetransformants was analyzed from culture supernatants of the 96 deep wellcultivations. Expression was verified by SDS-PAGE analysis using anE-Page 8% SDS-PAGE 48 well gel (Invitrogen, Carlsbad, Calif., USA) andCoomassie blue staining. Based on the level of expression by SDS-PAGE,one transformant was selected and designated Aspergillus oryzae CBH I.

For larger scale production, A. oryzae CBH I spores were spread ontoCOVE sucrose slants and incubated for five days at 37° C. The confluentspore slants were washed twice with 5 ml of 0.01% TWEEN® 20 to maximizethe number of spores collected. The spore suspensions were then used toinoculate seven 500 ml flasks containing 150 ml of DAP-4C medium. Thecultures were incubated at 30° C. with constant shaking at 100 rpm. Atday four post-inoculation, the culture broths were collected byfiltration through a bottle top MF75 Supor MachV 0.2 μm PES filter(Thermo Fisher Scientific, Roskilde, Denmark). Expression was verifiedby SDS-PAGE analysis using an E-Page 8% SDS-PAGE 48 well gel andCoomassie staining. The culture broths from A. oryzae CBH I produced aband at approximately 80 kDa for the T. reesei cellobiohydrolase I.

Example 6: Expression of the Trichoderma reesei Cellobiohydrolase I M6Variant

The expression plasmid pKHJN0059 was transformed into Aspergillus oryzaeMT3568 protoplasts according to Christensen et al., 1988, supra and WO2004/032648. A. oryzae MT3568 protoplasts were prepared according to themethod of European Patent, EP0238023, pages 14-15.

Transformants were purified on COVE sucrose plates through singleconidia prior to sporulating them on PDA plates. Spores of thetransformants were inoculated into 96 deep well plates containing 0.75ml of YP+2% glucose medium and incubated stationary at 30° C. for 4days. Production of the T. reesei cellobiohydrolase I M6 variant by thetransformants was analyzed from culture supernatants of the 96 deep wellcultivations. Expression was verified by SDS-PAGE analysis using anE-Page 8% SDS-PAGE 48 well gel and Coomassie staining. Based on thelevel of expression by SDS-PAGE, one transformant was selected forfurther work and designated Aspergillus oryzae M6.

For larger scale production, A. oryzae M6 spores were spread onto COVEsucrose slants and incubated for five days at 37° C. The confluent sporeslants were washed twice with 5 ml of 0.01% TWEEN® 20 to maximize thenumber of spores collected. The spore suspensions were then used toinoculate seven 500 ml flasks containing 150 ml of DAP-4C medium. Thecultures were incubated at 30° C. with constant shaking at 100 rpm. Atday four post-inoculation, the culture broths were collected byfiltration through a bottle top MF75 Supor MachV 0.2 μm PES filter.Expression was verified by SDS-PAGE analysis using an E-Page 8% SDS-PAGE48 well gel and Coomassie staining. The culture broths from A. oryzae M6produced a band at approximately 80 kDa for the T. reeseicellobiohydrolase I M6 variant.

Example 7: Purification of the Trichoderma reesei Wild-TypeCellobiohydrolase I and Cellobiohydrolase I M6 Variant

The filtered broths of A. oryzae CBH I (Example 5) and A. oryzae M6(Example 6) were adjusted to pH 7.0 and filtered using a 0.22 μm PESfilter (Nalge Nunc International Corp., Rochester, N.Y., USA). Thenammonium sulphate was added to each filtrate to a concentration of 1.8M.

Each filtrate was purified according to the following procedure. Thefiltrate was loaded onto a Phenyl SEPHAROSE® 6 Fast Flow column (highsub) (GE Healthcare, United Kingdom) equilibrated with 1.8 M ammoniumsulphate, 25 mM HEPES pH 7.0. After a wash with 0.54 M ammoniumsulphate, the bound proteins were batch eluted with 25 mM HEPES pH 7.0.Fractions were collected and analyzed by SDS-PAGE using 12-well NUPAGE®4-12% Bis-Tris gel (GE Healthcare, Piscataway, N.J., USA). The fractionswere pooled based on SDS-PAGE as above and applied to a SEPHADEX™ G-25(medium) column (GE Healthcare, United Kingdom) equilibrated with 25 mMMES pH 6.0. Fractions were collected, analyzed by SDS-PAGE as above, andpooled. The pooled fractions were applied to a 6 ml RESOURCE™ 15Q column(GE Healthcare, United Kingdom) equilibrated with 25 mM MES pH 6.0 andbound proteins were eluted with a linear 0-300 mM sodium chloridegradient (12 column volumes) for the wild-type cellobiohydrolase or alinear 0-350 mM sodium chloride gradient (14 column volumes) for thevariant. Fractions were collected and analyzed by SDS-PAGE, A₂₈₀, andactivity measurements using 4-nitrophenyl-beta-D-glucopyranoside (SigmaChemical Co., St. Louis, Mo., USA) and4-nitrophenyl-beta-D-lactopyranoside (Sigma Chemical Co., St. Louis,Mo., USA) as substrates. The assays were performed in 96-well Nuncmicrotiter plates (Thermo Scientific, Sunnyvale, Calif., USA). The assaybuffer was 50 mM Britton-Robinson buffer (50 mM H₃PO₄, 50 mM CH₃COOH, 50mM H₃BO₃) with 50 mM KCl, 1 mM CaCl₂, 0.01% TRITON® X-100, pH adjustedto 6.0 with NaOH. A 20 μl sample of protein solution was pipetted intoeach well and 120 μl of 1 mM substrate in the assay buffer were added.The substrate 4-nitrophenyl-beta-D-glucopyranoside was used to determinebeta-glucosidase activity and 4-nitrophenyl-beta-D-lactopyranoside forcellobiohydrolase I and cellobiohydrolase variant activity. A standardcurve was made by replacing the protein solution with 20 μl of4-nitrophenolate standard (0, 0.05, 0.075, 0.1, 0.2, 0.3, 0.4, 0.5 mM).If necessary, samples were diluted in the assay buffer to yieldabsorptions within the range of the standard curve. The plate was sealedand incubated in a thermomixer at 37° C. for 15 minutes with 750 rpmshaking. Immediately after incubation the reaction was stopped by adding100 μl of 0.5 M glycine-2 mM EDTA pH 10 and the absorption was measuredat 405 nm. The absorption of a “blank”, in which the protein was addedafter the stop solution, was recorded for each sample and subtractedfrom the result to obtain the absorption of released 4-nitrophenolate.

Based on SDS-PAGE, A₂₈₀, and the activity measurements, the fractionswere pooled to the final product.

The T. reesei wild-type cellobiohydrolase I and cellobiohydrolase I M6variant were purified to a concentration of 57 μM and 38 μM,respectively, as determined by A₂₈₀ using the calculated molarextinction coefficient 84810 M⁻¹·cm⁻¹ and 84810 M⁻¹·cm⁻¹, respectively.

Example 8: Activity Measurement on Microcrystalline Cellulose of theTrichoderma reesei Cellobiohydrolase M6 Variant

The activity of the purified cellobiohydrolase I M6 variant (Example 7)was compared to the purified T. reesei wild-type cellobiohydrolase I(Example 7) using microcrystalline cellulose (AVICEL® PH101;Sigma-Aldrich, St. Louis, Mo., USA) as a substrate. The microcrystallinecellulose was suspended at 60 g per liter of 2 mM CaCl₂−50 mM sodiumacetate pH 5 as assay buffer.

Activity of the T. reesei wild-type cellobiohydrolase I andcellobiohydrolase I M6 variant was measured in a water-jacketed glasscell connected to a Julabo F12 water bath (Buch & Holm A/S, Herlev,Denmark). Each reaction chamber was filled with 5 ml of themicrocrystalline cellulose suspension and magnetically stirred at 600rpm. The enzyme was injected into the cell using 250 μl glass syringes(Hamilton Co., Boston, Mass., USA) with a Fusion 100 syringe pump(Chemyx Inc., Stafford, Tex., USA) to a final concentration of 100 nM (5μg/ml) with an injection time of 1 second (wild-type: 8.8 μl, 528μl/minute; M6 variant: 13.16 μl, 789 μl/minute). The reactions wereallowed to proceed for 5 hours at 25° C. before being quenched with 80μl of 1 M NaOH.

From each reaction 2 samples of 2 ml were removed and filtered with a0.2 μM hydrophilic MINISART® NML syringe filter (Sartorius StedimBiotech S. A., Goettingen, Germany). The filtrates were diluted 1:10with milliQ water (control was measured undiluted) and the glucose,cellobiose, and cellotriose contents were analyzed using a DionexICS-5000 DC High-Performance Liquid Chromatography (HPLC) System (ThermoScientific, Sunnyvale, Calif., USA) equipped with a 4 mm×25 cm CARBOPAC™PA10 column (Thermo Scientific, Sunnyvale, Calif., USA), a Dionex GP40gradient pump (Thermo Scientific, Sunnyvale, Calif., USA), and a DionexED40 electrochemical detector (Thermo Scientific, Sunnyvale, Calif.,USA) with a gold working electrode (standard carbohydrate settings).Oligosaccharides were separated on the CARBOPAC™ PA10 column using thefollowing gradient program at a flow rate of 1 ml per minute: 0-4minutes isocratic elution with 50 mM sodium hydroxide; 4-28 minuteslinear gradient to 100 mM sodium acetate in 90 mM sodium hydroxide;28-29 minutes linear gradient to 450 mM sodium acetate in 200 mM sodiumhydroxide; 29-30 minutes linear gradient to 100 mM sodium hydroxide;30-31 minutes linear gradient to 50 mM sodium hydroxide; and 31-35minutes reequilibration under the initial conditions. Combined externalstandards were ([glucose]/[cellobiose]/[cellotriose]): 1 μM/2 μM/0.5 μM,2 μM/4 μM/1 μM, 3 μM/6 μM/1.5 μM, 4 μM/8 μM/2 μM, and 5 μM/10 μM/2.5 μM.Chromatogram peak integration, standard curve, and concentrationdetermination were performed using a CHROMELEON® 7 Chromatography DataSystem (Thermo Fisher Scientific, Roskilde, Denmark).

The results as shown in FIG. 1 and Table 1 demonstrated that thecellobiohydrolase I M6 variant had an approximately 65% increase inactivity toward microcrystalline cellulose compared to the wild-typecellobiohydrolase.

TABLE 1 Saccharide production from microcrystalline cellulose by the T.reesei wild-type cellobiohydrolase I and the cellobiohydrolase I M6variant thereof after 5 hours at pH 5 and 25° C. [glucose] (μM)[cellobiose] (μM) [cellotriose] (μM) Control 15.9 1.6 2.5 Wild-type 22.9± 0.3 121.4 ± 4.3 5.4 ± 4 × 10⁻³ M6 Variant 29.1 ± 10⁻³ 201.7 ± 0.5 8.1± 0.04

Example 9: Pretreated Corn Stover Hydrolysis Assay

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 1.4 wt % sulfuric acid at 165°C. and 107 psi for 8 minutes. The water-insoluble solids in thepretreated corn stover (PCS) contained 56.5% cellulose, 4.6%hemicellulose, and 28.4% lignin. Cellulose and hemicellulose weredetermined by a two-stage sulfuric acid hydrolysis with subsequentanalysis of sugars by high performance liquid chromatography using NRELStandard Analytical Procedure #002. Lignin was determinedgravimetrically after hydrolyzing the cellulose and hemicellulosefractions with sulfuric acid using NREL Standard Analytical Procedure#003.

Unmilled, unwashed PCS (whole slurry PCS) was prepared by adjusting thepH of the PCS to 5.0 by addition of 10 M NaOH with extensive mixing, andthen autoclaving for 20 minutes at 120° C. The dry weight of the wholeslurry PCS was 29%. Milled unwashed PCS (dry weight 32.35%) was preparedby milling whole slurry PCS in a Cosmos ICMG 40 wet multi-utilitygrinder (EssEmm Corporation, Tamil Nadu, India).

The hydrolysis of PCS was conducted using 2.2 ml deep-well plates(Axygen, Union City, Calif., USA) in a total reaction volume of 1.0 ml.The hydrolysis was performed with 50 mg of insoluble PCS solids per mlof 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfateand various protein loadings of various enzyme compositions (expressedas mg protein per gram of cellulose). Enzyme compositions were preparedand then added simultaneously to all wells in a volume ranging from 50μl to 200 μl, for a final volume of 1 ml in each reaction. The plate wasthen sealed using an ALPS300™ plate heat sealer (Abgene, Epsom, UnitedKingdom), mixed thoroughly, and incubated at a specific temperature for72 hours. All experiments reported were performed in triplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for sugar content as described below. When not usedimmediately, filtered aliquots were frozen at −20° C. The sugarconcentrations of samples diluted in 0.005 M H₂SO₄ were measured using a4.6×250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) by elution with 0.05% w/w benzoic acid-0.005 M H₂SO₄ at 65°C. at a flow rate of 0.6 ml per minute, and quantitation by integrationof the glucose, cellobiose, and xylose signals from refractive indexdetection (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies, SantaClara, Calif., USA) calibrated by pure sugar samples. The resultantglucose and cellobiose equivalents were used to calculate the percentageof cellulose conversion for each reaction.

Glucose and cellobiose were measured individually. Measured sugarconcentrations were adjusted for the appropriate dilution factor. Thenet concentrations of enzymatically-produced sugars from unwashed PCSwere determined by adjusting the measured sugar concentrations forcorresponding background sugar concentrations in unwashed PCS at zerotime point. All HPLC data processing was performed using MICROSOFTEXCEL™ software (Microsoft, Richland, Wash., USA).

The degree of cellulose conversion to glucose was calculated using thefollowing equation: % cellulose conversion=[glucoseconcentration+(1.053×cellobiose concentration)]/[(glucoseconcentration+(1.053×cellobiose concentration) in a limit digest]×100.In order to calculate % cellulose conversion, a 100% conversion pointwas set based on a cellulase control (100 mg of T. reesei cellulase pergram cellulose. Triplicate data points were averaged and standarddeviation was calculated.

Example 10: Preparation of an Enzyme Composition withoutCellobiohydrolase I

The Aspergillus fumigatus GH6A cellobiohydrolase II (SEQ ID NO: 32 [DNAsequence] and SEQ ID NO: 33 [deduced amino acid sequence]) was preparedrecombinantly in Aspergillus oryzae as described in WO 2011/057140. Thefiltered broth of the A. fumigatus cellobiohydrolase II was bufferexchanged into 50 mM sodium acetate pH 5.0 using a 400 ml SEPHADEX™ G-25column. The fractions were pooled.

The T. reesei GH5 endoglucanase II (SEQ ID NO: 34 [DNA sequence] and SEQID NO: 35 [deduced amino acid sequence]) was prepared recombinantlyaccording to WO 2011/057140 using Aspergillus oryzae as a host. Thefiltered broth of the T. reesei endoglucanase II was desalted andbuffer-exchanged into 20 mM Tris pH 8.0 using tangential flow (10Kmembrane, Pall Corporation).

The Penicillium sp. (emersonii) GH61A polypeptide (SEQ ID NO: 36 [DNAsequence] and SEQ ID NO: 37 [deduced amino acid sequence]) was preparedrecombinantly according to WO 2011/041397 using T. reesei as a host. Topurify P. emersonii GH61A polypeptide, a fermentation culture medium wasdesalted using a tangential flow concentrator (Pall Filtron,Northborough, Mass., USA) equipped with a 5 kDa polyethersulfonemembrane (Pall Filtron, Northborough, Mass., USA) into 20 mM Tris-HCl pH8.5. The buffer-exchanged sample was loaded onto a Q SEPHAROSE® FastFlow column (GE Healthcare, Piscataway, N.J., USA) preequilibrated with20 mM Tris-HCl, pH 8.0, eluted with 20 mM Tris-HCl pH 8.0 and 1 M NaCl.Selected fractions were pooled, made in 0.85 M ammonium sulfate, andloaded onto a Phenyl SEPHAROSE® Fast Flow column preequilibrated with 20mM Tris-HCl, pH 7.5 and 0.85 M ammonium sulfate, eluted with 20 mMTris-HCl, pH 7.5. The fractions were pooled and desalted using atangential flow concentrator (Pall Filtron, Northborough, Mass., USA)equipped with a 5 kDa polyethersulfone membrane into 50 mM sodiumacetate pH 5.0.

The Aspergillus fumigatus GH10 xylanase (xyn3) (SEQ ID NO: 38 [DNAsequence] and SEQ ID NO: 39 [deduced amino acid sequence]) was preparedrecombinantly according to WO 2006/078256 using Aspergillus oryzae BECh2(WO 2000/39322) as a host. The filtered broth of the A. fumigatusxylanase was desalted and buffer-exchanged into 50 mM sodium acetate pH5.0 using a HIPREP® 26/10 Desalting Column (GE Healthcare, Piscataway,N.J., USA).

The Aspergillus fumigatus Cel3A beta-glucosidase 4M mutant (SEQ ID NO:40 [DNA sequence] and SEQ ID NO: 41 [deduced amino acid sequence]) wasrecombinantly prepared according to WO 2012/044915. The filtered brothof Aspergillus fumigatus Cel3A beta-glucosidase 4M was concentrated andbuffer exchanged using a tangential flow concentrator (Pall Filtron,Northborough, Mass., USA) equipped with a 10 kDa polyethersulfonemembrane (Pall Filtron, Northborough, Mass., USA) with 50 mM sodiumacetate pH 5.0 containing 100 mM sodium chloride.

The Talaromyces emersonii CBS 393.64 beta-xylosidase (SEQ ID NO: 42 [DNAsequence] and SEQ ID NO: 43 [deduced amino acid sequence]) was preparedrecombinantly according to Rasmussen et al., 2006, Biotechnology andBioengineering 94: 869-876 using Aspergillus oryzae JaL355 as a host (WO2003/070956). The filtered broth was concentrated and desalted with 50mM sodium acetate pH 5.0 using a tangential flow concentrator equippedwith a 10 kDa polyethersulfone membrane.

The protein concentration for each of the monocomponents described abovewas determined using a Microplate BCA™ Protein Assay Kit (Thermo FischerScientific, Waltham, Mass., USA) in which bovine serum albumin was usedas a protein standard. An enzyme composition was prepared composed ofeach monocomponent as follows: 39.7% Aspergillus fumigatus Cel6Acellobiohydrolase II, 15.9% T. reesei GH5 endoglucanase II, 23.8%Penicillium sp. (emersonii) GH61A polypeptide, 7.9% Aspergillusfumigatus GH10 xylanase, 7.9% Aspergillus fumigatus beta-glucosidase,and 4.8% Talaromyces emersonii beta-xylosidase. The enzyme compositionis designated herein as “cellulolytic enzyme composition withoutcellobiohydrolase I”.

Example 11: Comparison of the Effect of the Trichoderma reeseiCellobiohydrolase I M6 Variant and Trichoderma reesei Wild-TypeCellobiohydrolase I in the Hydrolysis of Milled Unwashed PCS by aCellulase Enzyme Composition

The T. reesei cellobiohydrolase I M6 variant and T. reesei wild-typecellobiohydrolase I were added to the cellulolytic enzyme compositionwithout cellobiohydrolase I (Example 10) at 40° C. using milled unwashedPCS as a substrate. Each cellobiohydrolase I was added individually at0.8633, 1.295, and 1.9425 mg enzyme protein per g cellulose to 2.205 mgenzyme protein of the cellulase enzyme composition withoutcellobiohydrolase I per g cellulose.

The assay was performed as described in Example 9. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 24,48, and 72 hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mMmanganese sulfate. All reactions were performed in triplicate andinvolved single mixing at the beginning of hydrolysis.

The results shown in FIGS. 2, 3, and 4 demonstrated that at 24, 48, and72 hours, the cellulase enzyme composition that included the T. reeseicellobiohydrolase I M6 variant had significantly higher celluloseconversion than the cellulase enzyme composition that included T. reeseiwild-type cellobiohydrolase I.

Example 12: Determination of Td by Differential Scanning Calorimetry ofthe Trichoderma reesei Cellobiohydrolase I M6 Variant and Trichodermareesei Wild-Type Cellobiohydrolase I

The thermostability of the T. reesei wild-type cellobiohydrolase I andcellobiohydrolase I M6 variant was determined by Differential Scanningcalorimetry (DSC) using a VP-Capillary Differential Scanning calorimeter(MicroCal Inc., Piscataway, N.J., USA). The thermal denaturationtemperature, Td (° C.), was taken as the top of denaturation peak (majorendothermic peak) in thermograms (Cp vs. T) obtained after heatingenzyme solutions (approx. 1 mg/ml) in 50 mM sodium acetate pH 5.0 at aconstant programmed heating rate of 200 K/hour.

Sample- and reference-solutions (approx. 0.2 ml) were loaded into thecalorimeter (reference: buffer without enzyme) from storage conditionsat 10° C. and thermally pre-equilibrated for 20 minutes at 20° C. priorto DSC scan from 20° C. to 100° C. Denaturation temperatures weredetermined at an accuracy of approximately +/−1° C.

The results demonstrated that the T. reesei wild-type cellobiohydrolaseI has a Td of 69° C. compared to 68° C. for the cellobiohydrolase I M6variant thereof.

Example 13: Site-Directed Mutagenesis of the Wild-Type Trichodermareesei Cellobiohydrolase I

The codon-optimized synthetic gene encoding the wild-type T. reeseicellobiohydrolase I (Example 1) was used to generate the T. reeseicellobiohydrolase I TC1-111 variant (SEQ ID NO: 44 for the mutant DNAsequence and SEQ ID NO: 45 for the variant), an AAC codon (N198) wasreplaced with a GCA codon (198A).

To generate the T. reesei cellobiohydrolase I TC1-116 variant (SEQ IDNO: 46 for the mutant DNA sequence and SEQ ID NO: 47 for the variant), aGCC codon (A199) was deleted (A199*).

To generate the T. reesei cellobiohydrolase I TC1-61 variant (SEQ ID NO:48 for the mutant DNA sequence and SEQ ID NO: 49 for the variant), anAAC codon (N200) was replaced with a TGG codon (200W).

To generate the T. reesei cellobiohydrolase I TC1-103 variant (SEQ IDNO: 50 for the mutant DNA sequence and SEQ ID NO: 51 for the variant),an AAC codon (N200) was replaced with a GGA codon (200G).

Two synthetic primers for each site-directed mutagenesis were designedas shown below using an SOE primer design tool. The introducedsite-directed mutation changed an AAC codon (N198) to a GCA codon(198A), an AAC codon (N200) to a TGG codon (200W), and an AAC codon(N200) to a GGA codon (200G), and a GCC codon (A199) was deleted.

Primer F-N198A: (SEQ ID NO: 52)5′-GCTGGGAACCCTCGTCGAACGCAGCCAACACCGGCATTGGA-3′ Primer R-N198A:(SEQ ID NO: 53) 5′-GTTCGACGAGGGTTCCCAGCCTTCGACGTTTG-3′ Primer F-ΔA199:(SEQ ID NO: 54) 5′-TGGGAACCCTCGTCGAACAACAACACCGGCATTGGAGGCCAT-3′Primer R-ΔA199: (SEQ ID NO: 55) 5′-GTTGTTCGACGAGGGTTCCCAGCCTTCGACG-3′Primer F-N200W: (SEQ ID NO: 56)5′-GAACCCTCGTCGAACAACGCCTGGACCGGCATTGGAGGCCATGG-3′ Primer R-N200W:(SEQ ID NO: 57) 5′-GGCGTTGTTCGACGAGGGTTCCCAGCCTTCG-3′ Primer F-N200G:(SEQ ID NO: 58) 5′-GAACCCTCGTCGAACAACGCCGGAACCGGCATTGGAGGCCAT-3′Primer R-N200G: (SEQ ID NO: 59) 5′-GGCGTTGTTCGACGAGGGTTCCCAGCCTTCG-3′

Site-directed mutagenesis of the synthetic gene encoding the wild-typeT. reesei cellobiohydrolase was facilitated by PCR amplifications of thepDau109 vector containing the

T. reesei cellobiohydrolase I gene: The T. reesei cellobiohydrolase Igene was previously cloned into Bam HI-Hind III digested pDau109resulting in transcription of the T. reesei cellobiohydrolase I geneunder the control of a NA2-tpi double promoter. The mutations wereintroduced by PCR using a PHUSION® High-Fidelity PCR Kit. The PCRsolution was composed of 10 μl of 5×HF buffer, 1 μl of dNTPs (10 mM),0.5 μl of PHUSION® DNA polymerase (0.2 units/μl), 2.5 μl of primerF-N198A (10 μM), 2.5 μl of primer R-N198A (10 μM), 10 μl of template DNA(pDAu222—T. reesei cellobiohydrolase I, 1 ng/μl), and 23.5 μl ofdeionized water in a total volume of 50 μl. For the GCC deletion (A199*variant) 2.5 μl of primer F-AA199 (10 μM), 2.5 μl of primer R-AA199 (10μM) were used. For the ACC to TGG mutation (N200W variant) 2.5 μlprimer-F-N200W (10 μM) and 2.5 μl primer R-N200W (10 μM) were used. Forthe ACC to GGA mutation (N200G variant) 2.5 μl primer-F-N200G (10 μM)and 2.5 μl primer R-N200G (10 μM) were used.

The PCR was performed using a GeneAmp® PCR System 9700 (AppliedBiosystems, Foster City, Calif., USA) programmed for 1 cycle at 98° C.for 30 seconds; and 19 cycles each at 98° C. for 30 seconds, 55° C. for1 minute, and 72° C. for 4 minutes. The PCR solution was then held at15° C. until removed from the PCR machine.

Following PCR, 10 units of Dpn I were added directly to the PCR solutionand incubated at 37° C. for 1 hour. Then 1 μl of the Dpn I treated PCRsolution was transformed into ONE SHOT® TOP10F′ Chemically Competent E.coli cells according to the manufacturer's protocol and spread onto LBplates supplemented with 0.15 mg of ampicillin per ml. After incubationat 37° C. overnight, transformants were observed growing under selectionon the LB ampicillin plates. Two transformants were cultivated in LBmedium supplemented with 0.15 mg of ampicillin per ml and plasmids wereisolated using a QIAPREP® Spin Miniprep Kit.

The isolated plasmids were sequenced using an Applied Biosystems 3730xlDNA Analyzer with vector primers and T. reesei cellobiohydrolase 1 genespecific primers, shown below, in order to determine a representativeplasmid that was free of PCR errors and contained the desired mutations.

Primer F-pDau109 (SEQ ID NO: 30) 5′-CCCTTGTCGATGCGATGTATC-3′Primer F-Central1 (SEQ ID NO: 60) 5′-CATGTATCGTAAGCTCGCAGTCATCTCC-3′Primer F-Central2 (SEQ ID NO: 61) 5′-CTTCGTGTCGATGGACGCGG-3′Primer R-Central3 (SEQ ID NO: 62) 5′-GAACACGAGCCCCTCACTGC-3′Primer R-pDau109 (SEQ ID NO: 31) 5′-ATCCTCAATTCCGTCGGTCGA-3′

One plasmid clone free of PCR errors and containing the AAC (N198) toGCA (198A) mutation was chosen and designated plasmid pN198A.

One plasmid clone free of PCR errors and containing the deletion of theGCC codon (A199) to (A199*) mutation was chosen and designated plasmidpΔA199.

One plasmid clone free of PCR errors and containing the AAC (N200) toTGG (200VV) mutation was chosen and designated plasmid pN200W.

One plasmid clone free of PCR errors and containing the AAC (N200) toGGA (200G) mutation was chosen and designated plasmid pN200G.

pN198A was sequenced using primers F-Central1, F-Central2, R-Central3and R-pDau109. pΔA199 was sequenced using primers F-pDau109 F-Central1,F-Central2, R-Central3 and R-pDau109. pN200W was sequenced using primersF-Central1, F-Central2 and R-pDau109. pN200G was sequenced using primersF-Central1, F-Central2 and R-pDau109

Example 14: Expression of the Trichoderma reesei Cellobiohydrolase ITC1-111, TC1-116, TC1-61, and TC1-103 Variants

Expression of plasmids pN198A, pΔA199, pN200W, and pN200G in Aspergillusoryzae MT3568 was performed according to the protocol described inExample 6.

Expression was verified by SDS-PAGE analysis using an E-Page 8% SDS-PAGE48 well gel and Coomassie staining. Based on the level of expression bySDS-PAGE, one transformant was selected for each of plasmids pN198A,pΔA199, pN200W, and pN200G and designated Aspergillus oryzae→A199,N200G, N197A, and N200W, respectively.

For larger scale production, spores for each A. oryzae strain werespread onto COVE sucrose slants and incubated for five days at 37° C.Each confluent spore slant was washed twice with 5 ml of 0.01% TWEEN® 20to maximize the number of spores collected. Each spore suspensions werethen used to inoculate seven 500 ml flasks containing 150 ml of DAP-4Cmedium. The cultures were incubated at 30° C. with constant shaking at100 rpm. At day four post-inoculation, the culture broths were collectedby filtration through a bottle top MF75 Supor MachV 0.2 μm PES filter.Expression was verified by SDS-PAGE analysis using an E-Page 8% SDS-PAGE48 well gel and Coomassie staining. The culture broths from each A.oryzae strain produced a band at approximately 80 kDa for the T. reeseiA199*, N200G, N197A, and N200W cellobiohydrolase variant.

Example 16: Purification of the Trichoderma reesei Cellobiohydrolase ITC1-111, TC1-116, TC1-61, and TC1-103 Variants

The fermentation broths were filtered through PES Bottle top filter witha 0.22 μm cut-off (Thermo Fisher Scientific, Roskilde, Denmark).Ammonium sulphate was added to the filtered fermentation broths to makea 1.8 M solution.

The fermentation broths were purified by HIC/affinity chromatographyfollowed by IEX/affinity chromatography.

In the HIC/affinity chromatographic step, the fermentation broths wereapplied to a 200 ml Phenyl SEPHAROSE® 6 Fast Flow column (high sub)equilibrated with 1.8 M ammonium sulphate, 25 mM HEPES pH 7.0. Afterapplying the sample, the column was washed with 2 column volumes of 1.8M ammonium sulphate followed by 1 column volume of 0.54 M ammoniumsulphate. The bound proteins were batch eluted with 25 mM HEPES pH 7.0.

The elution of the protein was monitored at 280 nm. Fractions with high280 nm absorbance were analyzed by SDS-PAGE using 12-well NUPAGE® 4-12%Bis-Tris gel for their cellobiohydrolase content. Fractions with highcontent of this protein were pooled and collected for furtherpurification. The pooled fractions were desalted on a SEPHADEX™ G-25(medium) column equilibrated with 25 mM MES pH 6.0. The elution of theprotein was monitored at 280 nm and fractions with high absorbance at280 nm were chosen for the second chromatographic step.

The pooled fractions were applied to the 60 ml RESOURCE™ 15Q column(equilibrated with 25 mM MES pH 6.0 and bound proteins were eluted witha linear 50-300 mM sodium chloride gradient for 3 column volumes. Theelution of the protein was monitored at 280 nm and fractions with highabsorbance at 280 nm were analysed on SDS-PAGE.

Fractions with high content of cellobiohydrolase I were pooled.

Example 17: Activity Measurement on Microcrystalline Cellulose of theTrichoderma reesei Cellobiohydrolase I TC1-111, TC1-116, TC1-61, andTC1-103 Variants

The activity of the purified Trichoderma reesei cellobiohydrolase ITC1-111, TC1-116, TC1-61, and TC1-103 (variants were compared to thepurified T. reesei wild-type cellobiohydrolase I using washedmicrocrystalline cellulose (AVICEL® PH101; Sigma-Aldrich, St. Louis,Mo., USA) as a substrate.

The washed microcrystalline cellulose was prepared by applying andmixing (by hand) 180 g of microcrystalline cellulose and approximately400 ml of 0.22 μm filtered water to a centrifuge bottle (1 L). Thecentrifuge bottle was centrifuged at 4000 rpm for 5 minutes at 18° C.(Sorvall Hereaus Thermoscientific Sorvall Evolution RC superspeedcentrifuge). The supernatant was removed and 400 ml of MQ water wereadded again. This was repeated 4 times. At the 4^(th) repeat the pelletand 0.22 μm filtered water were mixed on a “Rocker” o/n (IKA KS 130basic) before centrifuging. The supernatant was removed and pellet wasre-suspended with 50 mM sodium acetate, 2 mM CaCl₂ pH 5 buffer to afinal concentration of 90 g/L.

The purified cellobiohydrolase variants were diluted in 50 mM sodiumacetate, 2 mM CaCl₂) pH 5 to a concentration of 9 μM. Then 100 μl of thediluted cellobiohydrolase I variants were added to each well of amicrotiter plate followed by 200 μl of washed microcrystalline celluloseat 90 g/liter to each well. The microtiter plate was quickly transferredto a thermomixer and incubated for 1 hour at 1100 rpm and 25° C. Thereaction was stopped by centrifugation at 3500 rpm for 3 minutes at 5°C. using a HERAEUS® MULTIFUGE® 3 s-r centrifuge (Thermo FisherScientific, Roskilde, Denmark). Fifty μl of supernatant were transferredto PCR sample tubes (0.2 ml non-skirtet 96-well PCR plate; Thermo FisherScientific, Roskilde, Denmark). PAHBAH (4-hydroxy-benzhydrazide) wasdissolved in buffer (0.18 M K-Na-tartrate and 0.5 M NaOH) to make a 15mg/ml solution. Seventy-five μl of the PAHBAH solution were added to thesupernatants in the PCR samples tubes.

The PCR sample tubes were placed in a Peltier Thermal Cycler andincubated at 95° C. for 10 minutes and 20° C. for 5 minutes. Afterincubation 100 μl were transferred to a 96 well microtiter plate and theabsorbance was measured at 410 nm. For each run a standard was included.The standard used was cellobiose diluted in 50 mM sodium acetate, 2 mMCaCl₂ pH 5 to a concentration of 0.008, 0.016, 0.0312, 0.0625, 0.125,0.25, 0.5, and 1 mM. In addition to the standard, a blank (withoutcellobiohydrolase) for each run was included. For all the measurements,the blank measurement was subtracted. The absorbance data werenormalized to cellobiose concentration using the standards.

The results as shown in FIG. 5 demonstrated that the cellobiohydrolaseA199* variant had an approximately 56% increase in activity towardmicrocrystalline cellulose compared to the wild-type cellobiohydrolaseI. N200G variant had 40%, N198A had 23% and N200W had 3% increase inactivity compared with wild-type cellobiohydrolase I.

Example 18: Pretreated Corn Stover Hydrolysis Assay

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) as described in Example 9.

Unmilled, unwashed PCS (whole slurry PCS) was prepared by adjusting thepH of the PCS to 5.0 by addition of 10 M NaOH with extensive mixing, andthen autoclaving for 20 minutes at 120° C. The dry weight of the wholeslurry PCS was 29%.

A 96-well plate was generated by machining a teflon plate of depth ¼inch with 96, cone-shaped wells, diameter ¼ inch at the upper surfaceand diameter ⅛ inch at the lower surface. The center of each well was atan equivalent position to the center of a corresponding well in astandard 96-well microtiter plate, approximately 23/64 inch on center.The resulting volume of each well was approximately 135 μl. This 96-wellaluminum plate is hereinafter referred to as the “fill plate”. ThepH-adjusted unmilled, unwashed PCS was used to fill the holes in thefill plate by applying a suitable volume of the PCS to the upper surfaceof the plate, then using a spatula to spread the material over thesurface and into the holes. Holes were deemed sufficiently full when thePCS was extruded through the hole in the bottom surface, formingnoodle-like tubes. A MULTISCREEN® Column Loader Scraper (Millipore,Billerica, Mass., USA) held perpendicular to the fill plate surface wasused to scrape excess PCS from the top and bottom surfaces of the fillplate, leaving the surfaces of the PCS in each well flush with thesurfaces of the fill plate. The fill plate was then placed on the top ofa 2.2 ml deep well plate (Axygen, Union City, Calif., USA) with the topsurface adjacent to the open end of the well plate (e.g., the top of thewell plate), and the wells aligned with the PCS-filled holes in the fillplate. The fill plate was secured in this position, and the assemblycentrifuged at 2500 rpm (1350×g) for 5 minutes in a Sorvall LegendRT+(Thermo Scientific, Waltham, Mass., USA). Following centrifugation,the PCS had been transferred to the deep well plate. A 3 mm glass bead(Fisher Scientific, Waltham, Mass., USA) was placed in each well formixing. The hydrolysis of PCS was conducted in a total reaction volumeof 0.2 ml. The hydrolysis was performed with 50 mg of insoluble PCSsolids containing 50 mM sodium acetate pH 5.0 buffer containing 1 mMmanganese sulfate and various protein loadings of various enzymecompositions (expressed as mg protein per gram of cellulose). Enzymecompositions were prepared and then added simultaneously to all wells ina volume ranging from 20 μl to 50 μl, for a final volume of 0.2-0.50 mlin each reaction. The plate was then sealed using an ALPS300™ plate heatsealer, mixed thoroughly, and incubated at a specific temperature for 72hours. All experiments reported were performed in triplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate and filtrates analyzed for sugar content asdescribed in Example 9.

Glucose was measured. The measured glucose concentration was adjustedfor the appropriate dilution factor. The net concentration ofenzymatically-produced glucose from unwashed PCS was determined byadjusting the measured glucose concentration for correspondingbackground glucose concentration in the unmilled, unwashed PCS at zerotime point. All HPLC data processing was performed using MICROSOFTEXCEL™ software.

The degree of glucose conversion to glucose was calculated using thefollowing equation: % glucose conversion=(glucoseconcentration)/(glucose concentration in a limit digest)×100. In orderto calculate % glucose conversion, a 100% conversion point was set basedon a cellulase control (100 mg of T. reesei cellulase per gramcellulose. Triplicate data points were averaged and standard deviationwas calculated.

Example 19: Preparation of an Enzyme Composition #2 withoutCellobiohydrolase I

An Aspergillus fumigatus GH6A cellobiohydrolase II variant(GENESEQP:AZN71803) was prepared recombinantly in Aspergillus oryzae asdescribed in WO 2011/123450. The filtered broth of the A. fumigatuscellobiohydrolase II was desalted and buffer-exchanged into 50 mM sodiumacetate pH 5.0 containing 100 mM sodium chloride using a tangential flow(10K membrane, Pall Corporation).

The Thermoascus aurantiacus GH5 endoglucanase II (GENESEQP:AZ104862) wasprepared recombinantly according to WO 2011/057140 using Aspergillusoryzae as a host. The filtered broth of the T. aurantiacus endoglucanaseII was concentrated using tangential flow (5K membrane, PallCorporation). The concentrated protein was desalted using a 400 ml

SEPHADEX™ G-25 column into 20 mM Tris pH 8.0. The Penicillium sp.(emersonii) GH61A polypeptide was prepared as disclosed in Example 10.

The Aspergillus fumigatus GH10 xylanase (xyn3) was prepared as disclosedin Example 10.

The Aspergillus fumigatus Cel3A beta-glucosidase 4M mutant was preparedas described in Example 10. The protein concentration was determinedusing a Microplate

BOA™ Protein Assay Kit (Thermo Fischer Scientific, Waltham, Mass., USA)in which bovine serum albumin was used as a protein standard.

The Talaromyces emersonii CBS 393.64 beta-xylosidase was prepared asdescribed in Example 10.

The protein concentration for each of the monocomponents described abovewas determined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard, except the Penicillium sp.(emersonii) GH61A polypeptide, which was determined at A280 using thetheoretical molar extinction coefficient 41730 M⁻¹·cm⁻¹. An enzymecomposition was prepared composed of each monocomponent as follows:39.7% Aspergillus fumigatus Cel6A variant cellobiohydrolase II, 15.9% T.reesei GH5 endoglucanase II, 23.8% Penicillium sp. (emersonii) GH61Apolypeptide, 7.9% Aspergillus fumigatus GH10 xylanase, 7.9% Aspergillusfumigatus beta-glucosidase, and 4.8% Talaromyces emersoniibeta-xylosidase. The enzyme composition is designated herein as“cellulolytic enzyme composition #2 without cellobiohydrolase I”.

Example 20: Source of DNA Sequence Information for Rasamsonia emersoniiCellobiohydrolase I

The genomic DNA sequence and deduced amino acid sequence of thewild-type Rasamsonia emersonii GH7 cellobiohydrolase I gene is shown inSEQ ID NO: 11 and SEQ ID NO: 12, respectively. The gene sequence is 99%identical to Genbank entry AF439935.4. The cDNA sequence and deducedamino acid sequence of the Rasamsonia emersonii cellobiohydrolase I geneis shown in SEQ ID NO: 63 and SEQ ID NO: 12, respectively.

Based on the cDNA sequence for Rasamsonia emersonii cellobiohydrolase I,a codon-optimized synthetic gene encoding the full-lengthcellobiohydrolase I was generated for Aspergillus oryzae expressionbased on the algorithm developed by Gustafsson et al., 2004, Trends inBiotechnology 22 (7): 346-353. The codon-optimized coding sequence (SEQID NO: 64) was synthesized by the GENEART® Gene Synthesis service (LifeTechnologies Corp., San Diego. Calif., USA) with a 5′ Bam HI restrictionsite, a 3′ Hind III restriction site, and a Kozac consensus sequence(CACC) situated between the start codon and the Bam HI restriction site.

Example 21: Site-Directed Mutagenesis of the Wild-Type Rasamsoniaemersonii Cellobiohydrolase I

The codon-optimized synthetic gene encoding the wild-type Rasamsoniaemersonii cellobiohydrolase I was provided in a non-specifiedkanamycin-resistant E. coli cloning vector.

To generate the R. emersonii cellobiohydrolase I PC1-146 variant (SEQ IDNO: 65 for the mutant DNA sequence and SEQ ID NO: 66 for the variant),an AAC codon (N194) was replaced with a GCA codon (194A) and an AACcodon (N197) was replaced with a GCA codon (197A).

Two synthetic primers for site-directed mutagenesis were designed asshown below using a SOE primer design tool. The introduced site-directedmutation changed an AAC codon (N194) to a GCA codon (194A) and an AACcodon (N197) to a GCA codon (197A).

Primer F-N194A N197A: (SEQ ID NO: 67)5′-AAGGATGGCAGCCCTCGTCCGCAAACGCGGCAACTGGCATCGGTGA TCAC-3′Primer R-N194A N197A: (SEQ ID NO: 68)5′-GGACGAGGGCTGCCATCCTTCCACGTTCGC-3′

Site-directed mutagenesis of the synthetic gene encoding the wild-typeR. emersonii cellobiohydrolase I was facilitated by PCR amplification ofthe pDau109 vector containing the R. emersonii cellobiohydrolase I genedesignated pDau222-R. emersonii cellobiohydrolase I. The R. emersoniicellobiohydrolase I gene was previously cloned into Bam HI-Hind IIIdigested pDau109 resulting in transcription of the R. emersoniicellobiohydrolase I gene under the control of a NA2-tpi double promoter.

The mutations were introduced by PCR using a PHUSION® High-Fidelity PCRKit. The PCR solution was composed of 10 μl of 5×HF buffer, 1 μl ofdNTPs (10 mM), 0.5 μl of PHUSION® DNA polymerase (0.2 units/μl), 0.25 μlof primer F-N194A N197A (100 μM), 0.25 μl of primer R-N194A N197A (100μM), 10 μl of template DNA (pDau222-R. emersonii cellobiohydrolase I, 1ng/μl), and 28 μl of deionized water in a total volume of 50 μl. The PCRwas performed using a GeneAmp® PCR System 9700 (Applied Biosystems,Foster City, Calif., USA) programmed for 1 cycle at 98° C. for 30seconds; and 19 cycles each at 98° C. for 30 seconds, 55° C. for 1minute, and 72° C. for 4 minutes. The PCR solution was then held at 8°C. until removed from the PCR machine.

Following PCR, 10 units of Dpn I were added directly to the PCR solutionand incubated at 37° C. for 1 hour. Then 1 μl of the Dpn I treated PCRsolution was transformed into ONE SHOT® TOP10F′ Chemically Competent E.coli cells according to the manufacturer's protocol and spread onto LBplates supplemented with 0.15 mg of ampicillin per ml. After incubationat 37° C. overnight, transformants were observed growing under selectionon the LB ampicillin plates. Four transformants were cultivated in LBmedium supplemented with 0.15 mg of ampicillin per ml and plasmids wereisolated using a QIAPREP® Spin Miniprep Kit.

The isolated plasmids were sequenced using an Applied Biosystems 3730xlDNA Analyzer with vector primers and R. emersonii cellobiohydrolase Igene specific primers, shown below, in order to determine arepresentative plasmid that was free of PCR errors and contained thedesired mutations.

Primer F-pDau109 (SEQ ID NO: 69) 5′-CCACACTTCTCTTCCTTCCTCAATCCTC-3′Primer F-Central1 (SEQ ID NO: 70) 5′-GTGAGGCGAACGTGGAAGGATG-3′Primer R-Central2 (SEQ ID NO: 71) 5′-gtacctgtgtccgtgccgtcatctg-3′Primer R-pDau109 (SEQ ID NO: 31) 5′-ATCCTCAATTCCGTCGGTCGA-3′

One plasmid clone free of PCR errors and containing the AAC (N194) toGCA (194A) mutation and the AAC (N197) to GCA (197A) mutation was chosenand designated plasmid pE146. The variant is designated herein asPC1-146.

Example 22: Construction of an Aspergillus oryzae Expression VectorContaining a Rasamsonia emersonii DNA Sequence EncodingCellobiohydrolase I

The kanamycin-resistant E. coli cloning vector provided by GENEART® GeneSynthesis encoding the Rasamsonia emersonii cellobiohydrolase I wasdigested with Fast Digest Bam HI and Hind III (Fermentas Inc., GlenBurnie, Md., USA) according to manufacturer's instructions. The reactionproducts were isolated by 1.0% agarose gel electrophoresis using TAEbuffer where a 1375 bp product band was excised from the gel andpurified using an ILLUSTRA™ GFX™ DNA Purification Kit.

The 1375 bp fragment was then cloned into pDau109 digested with Bam HIand Hind III using T4 DNA ligase. The Bam HI-Hind III digested pDau109and the Bam HI/Hind III fragment containing the T. reeseicellobiohydrolase I coding sequence were mixed in a molar ratio of 1:3(i.e., mass ratio approximately 2.5:1 or 20 ng:50 ng) and ligated with50 units of T4 DNA ligase in 1×T4 DNA ligase buffer with 1 mM ATP at 16°C. over-night in accordance with the manufacturer's instructions.Cloning of the Rasamsonia emersonii cellobiohydrolase I gene into BamHI-Hind III digested pDau109 resulted in the transcription of theRasamsonia emersonii cellobiohydrolase I gene under the control of aNA2-tpi double promoter.

The ligation mixture was transformed into ONE SHOT® TOP10F′ ChemicallyCompetent E. coli cells according to the manufacturer's protocol andspread onto LB plates supplemented with 0.1 mg of ampicillin per ml.After incubation at 37° C. overnight, transformants were observedgrowing under selection on the LB ampicillin plates. Insertion of theRasamsonia emersonii cellobiohydrolase I gene into pDau109 was verifiedby PCR on the transformants as described below using primers F-pDau109and R-pDau109.

A 1.1× REDDYMIX® Master Mix (Thermo Fisher Scientific, Roskilde,Denmark) was used for the PCR. The PCR solution was composed of 10 μl of1.1× REDDYMIX® Master Mix, 0.5 μl of primer F-pDau109 (10 μM), and 0.5μl of primer R-pDau109 (10 μM). A toothpick was used to transfer a smallamount of cells to the PCR solution. The PCR was performed using aPTC-200 DNA Engine programmed for 1 cycle at 94° C. for 3 minutes; 30cycles each at 94° C. for 30 seconds, 50° C. for 1 minute, and 72° C.for 2 minutes; and 1 cycle at 72° C. for 1 minute. The PCR solution wasthen held at 15° C. until removed from the PCR machine.

The PCR products were analyzed by 1.0% agarose gel electrophoresis usingTAE buffer where a 1600 bp PCR product band was observed confirminginsertion of the Rasamsonia emersonii cellobiohydrolase I codingsequence into pDau109.

An E. coli transformant containing the Rasamsonia emersoniicellobiohydrolase I plasmid construct was cultivated in LB mediumsupplemented with 0.1 mg of ampicillin per ml and plasmid DNA wasisolated using a QIAPREP® Spin Miniprep Kit. The plasmid was designatedpKHJN0135.

Example 23: Expression of the Wild-Type Rasamsonia emersoniiCellobiohydrolase I

The expression plasmid pKHJN0135 was transformed into Aspergillus oryzaeMT3568 protoplasts according to Christensen et al., 1988, supra and WO2004/032648. A. oryzae MT3568 protoplasts were prepared according to themethod of EP 0238023 B1, pages 14-15.

Transformants were purified on COVE sucrose plates through singleconidia prior to sporulating them on PDA plates. Spores of thetransformants were inoculated into 96 deep well plates containing 0.75ml of YP+2% glucose medium and incubated stationary at 30° C. for 4days. Production of the Rasamsonia emersonii cellobiohydrolase I by thetransformants was analyzed from culture supernatants of the 96 deep wellcultivations. Expression was verified by SDS-PAGE analysis using anE-Page 8% SDS-PAGE 48 well gel and Coomassie blue staining. Based on thelevel of expression by SDS-PAGE, one transformant was selected forfurther work and designated Aspergillus oryzae ReCBH I.

For larger scale production, A. oryzae ReCBH I spores were spread ontoCOVE sucrose slants and incubated for five days at 37° C. The confluentspore slants were washed twice with 5 ml of 0.01% TWEEN® 20 to maximizethe number of spores collected. The spore suspensions were then used toinoculate seven 500 ml flasks containing 150 ml of DAP-4C medium. Thecultures were incubated at 30° C. with constant shaking at 100 rpm. Atday four post-inoculation, the culture broths were collected byfiltration through a bottle top MF75 Supor MachV 0.2 μm PES filter.Expression was verified by SDS-PAGE analysis using an E-Page 8% SDS-PAGE48 well gel and Coomassie staining. The culture broths from A. oryzaeReCBH I produced a band at approximately 60 kDa for the Rasamsoniaemersonii cellobiohydrolase I.

For larger scale production, A. oryzae ReCBH I spores were spread ontoCOVE sucrose slants and incubated for five days at 37° C. The confluentspore slants were washed twice with 5 ml G2-Gly medium. The sporesuspensions were then used to inoculate 500 ml flasks containing 150 mlof G2-Gly medium. These pre-cultures were incubated at 30° C. withconstant shaking at 150 rpm. After one day, each of the pre-cultures wasused to inoculate four 500 ml flasks containing 150 ml DAP4C-1 medium.At day four post-inoculation, the culture broths were collected byfiltration through a bottle top M F75 Supor MachV 0.2 μm PES filter.

Example 24: Expression of the Rasamsonia emersonii Cellobiohydrolase IPC1-146 Variant

The expression plasmid pE146 was transformed into Aspergillus oryzaeMT3568 protoplasts according to Christensen et al., 1988, supra and WO2004/032648. A. oryzae MT3568 protoplasts were prepared according to themethod of EP 0238023 B1, pages 14-15.

Transformants were purified on COVE sucrose plates without CsCl throughsingle conidia. Spores of the transformants were inoculated into 96 deepwell plates containing 0.50 ml of YP+2% maltose medium and incubatedstationary at 34° C. for 6 days. Production of the R. emersoniicellobiohydrolase I PC1-146 variant by the transformants was analyzedfrom culture supernatants of the 96 deep well cultivations. Expressionwas verified by measuring released reducing sugars from hydrolysis ofmicrocrystalline cellulose. The hydrolysis was performed in 96 wellmicrotiter plates (NUNC Thermo Fisher Scientific, Roskilde, Denmark) at25° C. and 1100 rpm. Each hydrolysis reaction mixture contained 167 μlof microcrystalline cellulose at 90 g/liter in 50 mM sodium acetate pH5.0, 0.01% TRITON® X-100, 20 μl of culture supernatant, and 63 μl of 50mM sodium acetate pH 5.0, 0.01% TRITON® X-100. The plates were sealedwith tape. The hydrolysis reaction was stopped by spinning the plate at3500 rpm for 3 minutes. Then 50 μl of the reaction supernatant wereadded to 75 μl of stop solution in a 96 well PCR plate (Thermo FisherScientific, Roskilde, Denmark). The stop solution was composed of 15mg/ml 4-hydroxybenzhydrazide (Sigma Chemical Co., Inc., St. Louis, Mo.,USA), 50 mg/ml K-Na-tartrate (Sigma Chemical Co., Inc., St. Louis, Mo.,USA) in 2% (w/v) NaOH. The plate was sealed with a lid and the mixturewas incubated at 95° C. for 10 minutes and 5 minutes at 20° C. Then 100μl was transferred to a microtiter plate and absorbance at 410 nm wasmeasured using a SPECTRAMAX® Plus 384 (Molecular Devices, Sunnyvale,Calif., USA). The concentration of reducing sugar was proportional tothe absorbance at 410 nm of the oxidized 4-hydroxybenzhydrazide. Thereducing sugar content in the culture supernatants was measured byadding 4 μl of culture supernatant to a mixture of 75 μl of stopsolution and 46 μl of milliQ water in a 96 well PCR plate. The plate wassealed with a lid and the mixture was incubated at 95° C. for 10 minutesand 5 minutes at 20° C. Then 100 μl was transferred to a microtiterplate and the absorbance at 410 nm was measured. The absorbance at 410nm from the cell culture supernatant was subtracted from the absorbanceat 410 nm of the hydrolysis reaction, to measure the amount of reducingsugar released by the enzymes.

Based on the level of hydrolysis of the microcrystalline cellulose onetransformant was selected and designated A. oryzae PC1-146.

For larger scale production, A. oryzae PC1-146 spores were spread ontoCOVE sucrose slants and incubated for five days at 37° C. The confluentspore slants were washed twice with 5 ml of G2-Gly medium. The sporesuspensions were then used to inoculate 500 ml flasks containing 150 mlof G2-Gly medium. These pre-cultures were incubated at 30° C. withconstant shaking at 150 rpm. After one day, each of the pre-cultures wasused to inoculate four 500 ml flasks containing 150 ml of DAP-4C medium.At day four post-inoculation, the culture broths were collected byfiltration through a bottle top MF75 Supor MachV 0.2 μm PES filter.

Example 25: Purification of the Rasamsonia emersonii Wild-TypeCellobiohydrolase I and R. Emersonii Cellobiohydrolase I PC1-146 Variant

The fermentation broths were filtered through a PES Bottle top filterwith a 0.22 μm cut-off. Ammonium sulphate was added to the filteredfermentation broths to a concentration of 1.8 M. The fermentation brothswere purified by HIC/affinity chromatography followed by IEX/affinitychromatography.

In the HIC/affinity chromatographic step, the fermentation broths wereapplied to a 200 ml Phenyl SEPHAROSE® 6 Fast Flow column (high sub)equilibrated with 1.8 M ammonium sulphate, 25 mM HEPES pH 7.0. Afterapplying the sample, the column was washed with 2 column volumes of 1.8M ammonium sulphate followed by 1 column volume of 0.54 M ammoniumsulphate. The bound proteins were batch eluted with 25 mM HEPES pH 7.0.

The elution of the protein was monitored at 280 nm. Fractions with high280 nm absorbance were analyzed by SDS-PAGE using 12-well NUPAGE® 4-12%Bis-Tris gel for their cellobiohydrolase content. Fractions with highcontent of the protein were pooled and collected for furtherpurification. The pooled fractions were desalted on a SEPHADEX™ G-25(medium) column equilibrated with 25 mM MES pH 6.0. The elution of theprotein was monitored at 280 nm and fractions with high absorbance at280 nm were chosen for the second chromatographic step.

The pooled fractions were applied to a 60 ml RESOURCE™ 15Q columnequilibrated with 25 mM MES pH 6.0 and bound proteins were eluted with alinear 100-200 mM sodium chloride gradient for 1.5 column volumesfollowed by 1.5 column volumes of 300 mM sodium chloride, and followedby 1.5 column volumes of 1 M sodium chloride. The elution of the proteinwas monitored at 280 nm and fractions with high absorbance at 280 nmwere analyzed on SDS-PAGE. Fractions with high content ofcellobiohydrolase I were pooled.

Example 26: Activity Measurement on Microcrystalline Cellulose of theRasamsonia emersonii Cellobiohydrolase I PC1-146 Variant

The activity of the purified R. emersonii cellobiohydrolase I PC1-146variant was compared to the purified T. reesei wild-typecellobiohydrolase I using washed microcrystalline cellulose (AVICEL®PH101; Sigma-Aldrich, St. Louis, Mo., USA) as a substrate.

The purified cellobiohydrolase variant was diluted in 50 mM sodiumacetate, 2 mM CaCl₂ pH 5 to a concentration of 0.4 μM. Then 50 μl of thediluted cellobiohydrolase I variant were added to each well of amicrotiter plate followed by 200 μl of washed microcrystalline celluloseat 90 g/liter to each well. The microtiter plate was quickly transferredto a thermomixer and incubated for 1 hour at 1100 rpm and 50° C. Thereaction was stopped by centrifugation at 3500 rpm for 3 minutes at 5°C. using a HERAEUS® MULTIFUGE® 3 s-r centrifuge. Fifty μl of supernatantwere transferred to PCR sample tubes (0.2 ml non-skirtet 96-well PCRplate). PAHBAH (4-hydroxy-benzhydrazide) was dissolved in buffer (0.18 MK-Na-tartrate and 0.5 M NaOH) to make a 15 mg/ml solution. Seventy-fiveμl of the PAHBAH solution were added to the supernatants in the PCRsamples tubes.

The PCR sample tubes were placed in a Peltier Thermal Cycler andincubated at 95° C. for 10 minutes and 20° C. for 5 minutes. Afterincubation 100 μl were transferred to a 96 well microtiter plate and theabsorbance was measured at 410 nm. For each run a standard was included.The standard used was cellobiose diluted in 50 mM sodium acetate, 2 mMCaCl₂ pH 5 to a concentration of 0.008, 0.016, 0.0312, 0.0625, 0.125,0.25, 0.5, and 1 mM. In addition to the standard, a blank (withoutcellobiohydrolase) for each run was included. For all the measurements,the blank measurement was subtracted. The absorbance data werenormalized to cellobiose concentration using the standards.

The results as shown in FIG. 6 demonstrated that the R. emersoniicellobiohydrolase I PC1-146 variant had an approximately 17% increase inactivity toward microcrystalline cellulose compared to the R. emersoniiwild-type cellobiohydrolase I.

Example 27: Determination of Td of the Rasamsonia emersoniiCellobiohydrolase I PC1-146 Variant by Differential Scanning Calorimetry

The thermostability of the R. emersonii cellobiohydrolase I PC1-146variant was determined by Differential Scanning calorimetry (DSC) usinga VP-Capillary Differential Scanning calorimeter as described in Example12.

The results demonstrated that the R. emersonii wild-typecellobiohydrolase I has a Td of 78° C. compared to 78° C. for the R.emersonii cellobiohydrolase I PC1-146 variant thereof.

Example 28: Comparison of the Effect of Rasamsonia emersoniiCellobiohydrolase I PC1-146 Variant and Rasamsonia emersonii Wild-TypeCellobiohydrolase I on the Hydrolysis of Unwashed PCS by a CellulaseEnzyme Composition

The R. emersonii cellobiohydrolase I PC1-146 variant and R. emersoniiwild-type cellobiohydrolase I were added to cellulolytic enzymecomposition #2 without cellobiohydrolase I (Example 19) at 25° C. usingunmilled, unwashed PCS as a substrate. For hydrolysis at 8% totalsolids, each cellobiohydrolase I was added individually at 2.22 mgenzyme protein per g cellulose to 3.78 mg enzyme protein of thecellulase enzyme composition #2 without cellobiohydrolase I per gcellulose. For hydrolysis as 20% total solids, each cellobiohydrolase Iwas added individually at 4.44 mg enzyme protein per g cellulose to 7.56mg enzyme protein of the cellulase enzyme composition #2 withoutcellobiohydrolase I per g cellulose.

The assay was performed as described in Example 18. The reactions withunmilled, unwashed PCS (8% and 20% total solids) were conducted for 24,48, and 72 hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mMmanganese sulfate. All reactions were performed in quadruplicate andshaking at 200 rpm throughout the hydrolysis.

The results shown in FIG. 7 (8% total solids) and FIG. 8 (20% totalsolids) demonstrated that at 24, 48, and 72 hours, the cellulase enzymecomposition that included the R. emersonii cellobiohydrolase I PC1-146variant had significantly higher cellulose conversion than the cellulaseenzyme composition that included R. emersonii wild-typecellobiohydrolase I.

Example 29: Comparison of Rasamsonia emersonii Cellobiohydrolase IPC1-146 Variant with Rasamsonia emersonii Wild-Type Cellobiohydrolase IDuring Hydrolysis

Evaluation of the R. emersonii cellobiohydrolase I PC1-146 variant andthe R. emersonii wild-type cellobiohydrolase I was performed onunmilled, unwashed PCS at 20% TS. The enzyme matrix design is shownbelow in Table 1. CELLIC® HTec3 was obtained from Novozymes A/S,Bagsvaerd, Denmark. Enzymatic hydrolysis was conducted at 32° C. for 3and 5 days. The sugar released was analyzed by HPLC (1200 Series LCSystem, Agilent Technologies Inc., Palo Alto, Calif., USA) equipped witha Rezex ROA-Organic acid H⁺ column (8%) (7.8×300 mm) (Phenomenex Inc.,Torrance, Calif., USA), 0.2 mm in line filter, an automated sampler, agradient pump and a refractive index detector. The mobile phase used was5 mM sulfuric acid at a flow rate of 0.9 ml/min. Glucose at differentconcentrations was used as standards.

The results as shown in FIG. 9 demonstrated that the R. emersoniicellobiohydrolase I PC1-146 variant performed better than the wild-typeR. emersonii cellobiohydrolase I.

TABLE 1 enzyme dose R. emersonii R. emersonii mg/g CBH I CBH I CELLIC ®Tube glucan Variant Wild-type Af CBH II Ta EG Aa BG Htec3 1 6 37.5%37.5% 10% 5% 10% 2 6 37.5% 37.5% 10% 5% 10%

Example 30: Comparison of Rasamsonia emersonii Cellobiohydrolase IPC1-146 Variant with Rasamsonia emersonii Wild-Type Cellobiohydrolase IDuring Simultaneous Saccharification and Fermentation (SSF)

The experimental design was the same as shown in Example 29, except thatRED STAR® yeast at 1 g per liter and urea at 2 g per liter were addedtogether with the enzyme during the beginning of the hydrolysis. Ethanolrelease was analyzed by HPLC using the system described in Example 29.Ethanol at different concentrations was used as standards.

The results as shown in FIG. 10 demonstrate that the R. emersoniicellobiohydrolase I PC1-146 variant performed better than the wild-typeR. emersonii cellobiohydrolase I during SSF.

Example 31: Construction of a Rasamsonia emersonii FusionCellobiohydrolase I with Linker and Carbohydrate Binding Module fromTrichoderma reesei Cellobiohydrolase I

The codon-optimized synthetic gene encoding the T. reesei (H. jecorina)cellobiohydrolase I is described in Example 1.

The codon-optimized synthetic gene encoding the R. emersoniicellobiohydrolase I is described in Example 20.

To generate a gene encoding a R. emersonii fusion cellobiohydrolase Iwith linker and carbohydrate binding module (CBM) from T. reeseicellobiohydrolase I (SEQ ID NO: 72 for the fusion protein DNA sequenceand SEQ ID NO: 73 for the fusion protein), a DNA fragment encoding T.reesei cellobiohydrolase I linker and CBM was assembled to the 3′-end ofthe gene encoding the R. emersonii cellobiohydrolase I using splicingoverlap extension (SOE) PCR.

The DNA fragment encoding the T. reesei cellobiohydrolase I linker andCBM was amplified using primer F-SOE and primer R-pDau109 shown below.

Primer F-SOE (SEQ ID NO: 74)5′-GGTCCCATCAACTCGACATTCACAGCCTCGGGTGGAAACCCTCCTGG CGGAAACCCTC-3′Primer R-pDau109 (SEQ ID NO: 31) 5′-ATCCTCAATTCCGTCGGTCGA-3′Primer F-pDau109 (SEQ ID NO: 69) 5′-CCACACTTCTCTTCCTTCCTCAATCCTC-3′

The amplification of the DNA fragment encoding the T. reeseicellobiohydrolase 1 linker and CBM was performed using a PHUSION®High-Fidelity PCR Kit. The PCR solution was composed of 10 μl of 5×HFbuffer, 1 μl of dNTPs (10 mM), 0.5 μl of PHUSION® DNA polymerase (0.2units/μl), 0.25 μl of primer F-SOE (100 μM), 0.25 μl of primer R-pDau109(100 μM), 10 μl of template DNA (pDAu222-T. reesei cellobiohydrolase I,1 ng/μl), and 28 μl of deionized water in a total volume of 50 μl. ThePCR was performed using a GeneAmp® PCR System 9700 programmed for 1cycle at 98° C. for 30 seconds; and 30 cycles each at 98° C. for 10seconds, 55° C. for 30 seconds, and 72° C. for 1 minute. The PCRsolution was then held at 8° C. until removed from the PCR machine.

The PCR solution was submitted to 1% agarose gel electrophoresis usingTAE buffer where a 405 bp PCR fragment encoding the T. reesei a linkerand CBM was excised from the gel and purified using a MinElute GelExtraction Kit (QIAGEN Inc., Valencia, Calif., USA).

A DNA fragment encoding the R. emersonii cellobiohydrolase I wasamplified using primer F-pDau109 and primer R-pDau109 above.

The amplification of the DNA fragment encoding the R. emersoniiwild-type cellobiohydrolase I was performed using a PHUSION®High-Fidelity PCR Kit. The PCR solution was composed of 10 μl of 5×HFbuffer, 1 μl of dNTPs (10 mM), 0.5 μl of PHUSION® DNA polymerase (0.2units/μl), 0.25 μl of primer F-pDAu109 (100 μM), 0.25 μl of primerR-pDau109 (100 μM), 10 μl of template DNA (pDAu222-R. emersoniicellobiohydrolase I, 1 ng/μl), and 28 μl of deionized water in a totalvolume of 50 μl. The PCR was performed using a GeneAmp® PCR System 9700programmed for 1 cycle at 98° C. for 30 seconds; and 30 cycles each at98° C. for 10 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute.The PCR solution was then held at 8° C. until removed from the PCRmachine.

The PCR solution was submitted to 1% agarose gel electrophoresis usingTAE buffer where a 1600 bp fragment encoding the R. emersonii wild-typecellobiohydrolase I was excised from the gel and purified using aMinElute Gel Extraction Kit.

The two purified DNA fragments were assembled using SOE PCR and aPHUSION® High-Fidelity PCR Kit. The PCR solution was composed of 10 μlof 5×HF buffer, 1 μl of dNTPs (10 mM), 0.5 μl of PHUSION® DNA polymerase(0.2 units/μl), 0.25 μl of primer F-pDAu109 (100 μM), 10 μl of gelpurified fragment encoding T. reesei cellobiohydrolase 1 linker and CBM,2 μl of DNA fragment encoding R. emersonii cellobiohydrolase I, and 26μl of deionized water in a total volume of 50 μl. The PCR was performedusing a GeneAmp® PCR System 9700 programmed for 1 cycle at 98° C. for 30seconds; and 30 cycles each at 98° C. for 20 seconds, 55° C. for 30seconds, and 72° C. for 1 minute. The PCR solution was then held at 8°C. until removed from the PCR machine.

The PCR generated DNA fragment was then digested with Bam HI (NewEngland Biolabs, Ipswich, Mass., USA) and Hind III (New England Biolabs,Ipswich, Mass., USA) as follows. Forty μl of PCR product were mixed with5 μl buffer 2 (New England Biolabs, Ipswich, Mass., USA), 1 μl of BamHI, and 1 μl of Hind III and incubated for 4 hours at 37° C. Theresulting DNA product was submitted to 1% agarose gel electrophoresisusing TAE buffer. A band of approximately 1567 bp was excised from thegel and purified using a MinElute Gel Extraction Kit.

The purified 1567 bp fragment encoding the R. emersoniicellobiohydrolase I with linker and carbohydrate binding module (CBM)from T. reesei cellobiohydrolase I was cloned into pDAu109 digested withBam HI and Hind III using T4 DNA ligase. The Bam HI-Hind III digestedpDau109 and the Bam HI/Hind III fragment containing the R. emersoniicellobiohydrolase I with linker and carbohydrate binding module (CBM)from T. reesei cellobiohydrolase I coding sequence were mixed in a molarratio of 1:3 (i.e., equal volumes of gel purified products) and ligatedwith 50 units of T4 DNA ligase in 1×T4 DNA ligase buffer with 1 mM ATPand incubated at 22° C. for 10 minutes.

The ligation mixture was transformed into ONE SHOT® TOP10F′ ChemicallyCompetent E. coli cells according to the manufacturer's protocol andspread onto LB plates supplemented with 0.1 mg of ampicillin per ml.After incubation at 37° C. overnight, transformants were observedgrowing under selection on the LB ampicillin plates. Two transformantswere cultivated in LB medium supplemented with 0.15 mg of ampicillin perml and plasmids were isolated using a QIAPREP® Spin Miniprep Kit.

The insertion of the DNA fragment encoding the R. emersoniicellobiohydrolase I with linker and carbohydrate binding module (CBM)from T. reesei cellobiohydrolase I into pDAu109 was verified bysequencing. The isolated plasmids were sequenced using an AppliedBiosystems 3730xl DNA Analyzer with vector primers F-pDau109 andR-pDau109 in order to determine a representative plasmid that was freeof PCR errors and contained the correct insertion.

One plasmid clone free of PCR errors and containing the DNA fragmentencoding the R. emersonii cellobiohydrolase I with linker andcarbohydrate binding module (CBM) from T. reesei cellobiohydrolase I waschosen and designated plasmid pE147. The fusion cellobiohydrolase I isdesignated herein as PC1-147.

Example 32: Site-Directed Mutagenesis of the Rasamsonia emersoniiPC1-147 Fusion Cellobiohydrolase I

The fusion protein gene encoding the R. emersonii PC1-147 fusioncellobiohydrolase I was provided in pE147.

To generate a variant of the R. emersonii PC1-147 fusioncellobiohydrolase I (SEQ ID NO: 75 for the mutant DNA sequence and SEQID NO: 76 for the variant), an AAC codon (N194) was replaced with a GCAcodon (194A) and an AAC codon (N197) was replaced with a GCA codon(197A) in the gene encoding the R. emersonii PC1-147 fusioncellobiohydrolase

Two synthetic primers for site-directed mutagenesis were designed asshown below using a SOE primer design tool. The introduced site-directedmutation changed an AAC codon (N194) to a GCA codon (194A) and an AACcodon (N197) to a GCA codon (197A).

Primer F-N194A N197A: (SEQ ID NO: 67)5′-AAGGATGGCAGCCCTCGTCCGCAAACGCGGCAACTGGCATCGGTGA TCAC-3′Primer R-N194A N197A: (SEQ ID NO: 68)5′-GGACGAGGGCTGCCATCCTTCCACGTTCGC-3′

Site-directed mutagenesis of the R. emersonii PC1-147 fusioncellobiohydrolase I gene was facilitated by PCR amplifications of thepDau109 vector containing the R. emersonii PC1-147 fusioncellobiohydrolase I gene. The R. emersonii PC1-147 fusioncellobiohydrolase I gene was previously cloned into Bam HI-Hind IIIdigested pDau109 resulting in transcription of the R. emersonii PC1-147fusion cellobiohydrolase I gene under the control of a NA2-tpi doublepromoter.

The mutations were introduced by PCR using a PHUSION® High-Fidelity PCRKit. The PCR solution was composed of 10 μl of 5×HF buffer, 1 μl ofdNTPs (10 mM), 0.5 μl of PHUSION® DNA polymerase (0.2 units/μl), 0.25 μlof primer F-N194A N197A (100 μM), 0.25 μl of primer R-N194A N197A (100μM), 10 μl of plasmid pE147 DNA (1 ng/μl), and 28 μl of deionized waterin a total volume of 50 μl. The PCR was performed using a GeneAmp® PCRSystem 9700 programmed for 1 cycle at 98° C. for 30 seconds; and 19cycles each at 98° C. for 30 seconds, 55° C. for 1 minute, and 72° C.for 4 minutes. The PCR solution was then held at 8° C. until removedfrom the PCR machine.

Following the PCR, 10 units of Dpn I were added directly to the PCRsolution and incubated at 37° C. for 1 hour. Then 1 μl of the Dpn Itreated PCR solution was transformed into ONE SHOT® TOP10F′ ChemicallyCompetent E. coli cells according to the manufacturer's protocol andspread onto LB plates supplemented with 0.15 mg of ampicillin per ml.After incubation at 37° C. overnight, transformants were observedgrowing under selection on the LB ampicillin plates. Four transformantswere cultivated in LB medium supplemented with 0.15 mg of ampicillin perml and plasmids were isolated using a QIAPREP® Spin Miniprep Kit.

The isolated plasmids were sequenced using an Applied Biosystems 3730xlDNA Analyzer with primers F-pDau109, F-Central1, R-Central2 andR-pDau109, in order to determine a representative plasmid that was freeof PCR errors and contained the desired mutations.

Primer F-pDau109 (SEQ ID NO: 69) 5′-CCACACTTCTCTTCCTTCCTCAATCCTC-3′Primer F-Central1 (SEQ ID NO: 70) 5′-GTGAGGCGAACGTGGAAGGATG-3′Primer R-Central2 (SEQ ID NO: 71) 5′-gtacctgtgtccgtgccgtcatctg-3′Primer R-pDau109 (SEQ ID NO: 31) 5′-ATCCTCAATTCCGTCGGTCGA-3′

One plasmid clone free of PCR errors and containing the AAC (N194) toGCA (194A) mutation and the AAC (N197) to GCA (197A) mutation was chosenand designated plasmid pE378. The variant is designated herein asPC1-378.

Example 33: Expression of the Rasamsonia emersonii PC1-147 FusionCellobiohydrolase I and R. Emersonii Cellobiohydrolase I PC1-378 Variant

The expression plasmids pE147 and pE378 were transformed intoAspergillus oryzae MT3568 protoplasts according to Christensen et al.,1988, supra and WO 2004/032648. A. oryzae MT3568 protoplasts wereprepared according to the method of EP 0238023 B1, pages 14-15.

Transformants were purified on COVE sucrose plates without CsCl throughsingle conidia. Spores of the transformants were inoculated into 96 deepwell plates containing 0.50 ml of YP+2% maltose+0.5% glucose medium andincubated stationary at 34° C. for 6 days. Production of the R.emersonii PC1-147 fusion cellobiohydrolase I and the R. emersoniicellobiohydrolase I PC1-378 variant by the transformants were analyzedfrom culture supernatants of the 96 deep well cultivations. Expressionwas verified by measuring the released reducing sugars from hydrolysisof microcrystalline cellulose according to the procedure described inExample 24.

Based on the level of hydrolysis of the microcrystalline cellulose onetransformant for the R. emersonii PC1-147 fusion cellobiohydrolase I andthe R. emersonii cellobiohydrolase I PC1-378 variant were selected anddesignated A. oryzae PC1-147 and A. oryzae PC1-378, respectively.

For larger scale production, A. oryzae PC1-147 or A. oryzae PC1-378spores were spread onto COVE sucrose slants and incubated for five daysat 37° C. The confluent spore slants were washed twice with 5 ml ofG2-Gly medium. The spore suspensions were then used to inoculate 500 mlflasks containing 150 ml of G2-Gly medium. These pre-cultures wereincubated at 30° C. with constant shaking at 150 rpm. After one day,each of the pre-cultures was used to inoculate four 500 ml flaskscontaining 150 ml of DAP-4C medium. At day four post-inoculation, theculture broths were collected by filtration through a bottle top MF75Supor MachV 0.2 μm PES filter.

Example 34: Purification of the Rasamsonia emersonii PC1-147 FusionCellobiohydrolase I and R. Emersonii Cellobiohydrolase I PC1-378 Variant

The fermentation broths were filtered through a PES Bottle top filterwith a 0.22 μm cut-off. Ammonium sulphate was added to the filteredfermentation broths to a concentration of 1.8 M.

The fermentation broths were purified by HIC/affinity chromatographyfollowed by IEX/affinity chromatography.

In the HIC/affinity chromatographic step, the fermentation broths wereapplied to a 200 ml Phenyl SEPHAROSE® 6 Fast Flow column (high sub)which had been pre-equilibrated with 1.8 M ammonium sulphate, 25 mMHEPES pH 7.0. After applying the sample, the column was washed with 2column volumes of 1.8 M ammonium sulphate followed by 1 column volume of0.54 M ammonium sulphate. The bound proteins were batch eluted with 25mM HEPES pH 7.0.

The elution of the protein was monitored at 280 nm. Fractions with high280 nm absorbance were analysed on SDS-PAGE using 12-well NUPAGE® 4-12%Bis-Tris gel for their cellobiohydrolase I content. Fractions with highcontent of this protein were pooled and collected for furtherpurification. The pooled fractions were desalted on a SEPHADEX™ G-25(medium) column equilibrated with 25 mM MES pH 6.0. The elution of theprotein was monitored at 280 nm and fractions with high absorbance at280 nm were chosen for the second chromatographic step.

The pooled fractions were applied to the 60 ml RESOURCE™ 15Q columnequilibrated with 25 mM MES pH 6.0 and bound proteins were eluted with alinear 100-200 mM sodium chloride gradient for 1.5 column volumesfollowed by 1.5 column volumes of 300 mM sodium chloride, followed by1.5 column volumes of 1 M sodium chloride. The elution of the proteinwas monitored at 280 nm and fractions with high absorbance at 280 nmwere analysed on SDS-PAGE.

Fractions with high content of cellobiohydrolase I were pooled.

Example 35: Activity Measurement on Microcrystalline Cellulose of theRasamsonia emersonii PC1-147 Fusion Cellobiohydrolase I and the R.emersonii Cellobiohydrolase I

PC1-378 Variant

The activity of the purified R. emersonii PC1-147 fusioncellobiohydrolase I and the R. emersonii cellobiohydrolase I PC1-378variant (Example 34) were compared to the purified wild-type R.emersonii cellobiohydrolase I (Example 25) using washed microcrystallinecellulose as a substrate according to Example 26. Values are shown inrelative activity where 100% was set as the activity of R. emersoniiwild-type cellobiohydrolase I. The assay conditions were 24 hoursincubation at pH 5, 50° C. and 1100 rpm.

The results as shown in FIG. 11 demonstrated that the R. emersoniiPC1-147 fusion cellobiohydrolase I and the R. emersoniicellobiohydrolase I PC1-378 variant had an approximately 22% and 44%increase in activity toward microcrystalline cellulose, respectively,compared to the R. emersonii wild-type cellobiohydrolase I.

The inventions are further described by the following numberedparagraphs:

[1] A cellobiohydrolase variant, comprising an alteration at one or morepositions corresponding to positions 197, 198, 199, and 200 of themature polypeptide of SEQ ID NO: 2, wherein the alteration at the one ormore positions corresponding to positions 197, 198, and 200 is asubstitution and the alteration at the position corresponding toposition 199 is a deletion, wherein the variant has cellobiohydrolaseactivity, and wherein the variant has at least 60%, e.g., at least 65%,at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100%, sequenceidentity to the mature polypeptide of a parent cellobiohydrolase.

[2] The variant of paragraph 1, wherein the parent cellobiohydrolase isselected from the group consisting of: (a) a polypeptide having at least60% sequence identity to the mature polypeptide of SEQ ID NO: 2, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22; (b) a polypeptide encoded bya polynucleotide that hybridizes under low stringency conditions withthe mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 21; or thefull-length complement thereof; (c) a polypeptide encoded by apolynucleotide having at least 60% identity to the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, or SEQ ID NO: 21; and (d) a fragment of the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ IDNO: 22, which has cellobiohydrolase activity.

[3] The variant of paragraph 2, wherein the parent cellobiohydrolase hasat least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity to the mature polypeptide of SEQ ID NO: 2, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22.

[4] The variant of paragraph 2 or 3, wherein the parentcellobiohydrolase is encoded by a polynucleotide that hybridizes underlow stringency conditions, medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with the mature polypeptide coding sequence of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, orSEQ ID NO: 21; or the full-length complement thereof.

[5] The variant of any of paragraphs 2-4, wherein the parentcellobiohydrolase is encoded by a polynucleotide having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO:21.

[6] The variant of any of paragraphs 2-5, wherein the parentcellobiohydrolase comprises or consists of the mature polypeptide of SEQID NO: 2, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22.

[7] The variant of any of paragraphs 2-6, wherein the parentcellobiohydrolase is a fragment of the mature polypeptide of SEQ ID NO:2, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22, wherein the fragmenthas cellobiohydrolase activity.

[8] The variant of any of paragraphs 1-7, which has at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%, butless than 100%, sequence identity to the amino acid sequence of theparent cellobiohydrolase or the mature polypeptide thereof.

[9] The variant of any of paragraphs 1-8, wherein the number ofalterations is 1-4, e.g., 1, 2, 3, and 4 alterations.

[10] The variant of any of paragraphs 1-9, which comprises asubstitution at a position corresponding to position 197.

[11] The variant of paragraph 10, wherein the substitution is with Ala.

[12] The variant of any of paragraphs 1-11, which comprises asubstitution at a position corresponding to position 198.

[13] The variant of paragraph 12, wherein the substitution is with Ala.

[14] The variant of any of paragraphs 1-13, which comprises asubstitution at a position corresponding to position 200.

[15] The variant of paragraph 14, wherein the substitution is with Ala,Gly, or Trp.

[16] The variant of any of paragraphs 1-15, which comprises a deletionat a position corresponding to position 197.

[17] The variant of any of paragraphs 1-16, which comprises analteration at two positions corresponding to any of positions 197, 198,199, and 200.

[18] The variant of any of paragraphs 1-16, which comprises analteration at three positions corresponding to any of positions 197,198, 199, and 200.

[19] The variant of any of paragraphs 1-16, which comprises analteration at each position corresponding to positions 197, 198, 199,and 200.

[20] The variant of any of paragraphs 1-19, which comprises one or morealterations selected from the group consisting of N197A, N198A, A199*,and N200A,G,W at positions corresponding to the mature polypeptide ofSEQ ID NO: 2.

[21] The variant of any of paragraphs 1-19, which comprises thealterations N197A+N198A at positions corresponding to the maturepolypeptide of SEQ ID NO: 2.

[22] The variant of any of paragraphs 1-19, which comprises thealterations N197A+A199* at positions corresponding to the maturepolypeptide of SEQ ID NO: 2.

[23] The variant of any of paragraphs 1-19, which comprises thealterations N197A+N200A,G,W at positions corresponding to the maturepolypeptide of SEQ ID NO: 2.

[24] The variant of any of paragraphs 1-19, which comprises thealterations N198A+A199* at positions corresponding to the maturepolypeptide at positions corresponding to SEQ ID NO: 2.

[25] The variant of any of paragraphs 1-19, which comprises thealterations N198A+N200A,G,W at positions corresponding to the maturepolypeptide of SEQ ID NO: 2.

[26] The variant of any of paragraphs 1-19, which comprises thealterations A199*+N200A,G,W at positions corresponding to the maturepolypeptide of SEQ ID NO: 2.

[27] The variant of any of paragraphs 1-19, which comprises thealterations N197A+N198A+A199* at positions corresponding to the maturepolypeptide of SEQ ID NO: 2.

[28] The variant of any of paragraphs 1-19, which comprises thealterations N197A+N198A+N200A,G,W at positions corresponding to themature polypeptide of SEQ ID NO: 2.

[29] The variant of any of paragraphs 1-19, which comprises thealterations N197A+A199*+N200A,G,W at positions corresponding to themature polypeptide of SEQ ID NO: 2.

[30] The variant of any of paragraphs 1-19, which comprises thealterations N198A+

A199*+N200A,G,W at positions corresponding to the mature polypeptide ofSEQ ID NO: 2.

[31] The variant of any of paragraphs 1-19, which comprises thealterations N197A+N198A+A199*+N200A,G,W at positions corresponding tothe mature polypeptide of SEQ ID NO: 2.

[32] The variant of any of paragraphs 1-31, which comprises or consistsof SEQ ID NO: 6 or the mature polypeptide thereof.

[33] The variant of any of paragraphs 1-31, which comprises or consistsof SEQ ID NO: 45 or the mature polypeptide thereof.

[34] The variant of any of paragraphs 1-31, which comprises or consistsof SEQ ID NO: 47 or the mature polypeptide thereof.

[35] The variant of any of paragraphs 1-31, which comprises or consistsof SEQ ID NO: 49 or the mature polypeptide thereof.

[36] The variant of any of paragraphs 1-31, which comprises or consistsof SEQ ID NO: 51 or the mature polypeptide thereof.

[37] The variant of any of paragraphs 1-31, which comprises or consistsof SEQ ID NO: 66 or the mature polypeptide thereof.

[38] The variant of any of paragraphs 1-31, wherein the parent is ahybrid or chimeric polypeptide in which the carbohydrate binding domainof the parent is replaced with a different carbohydrate binding domain.

[39] The variant of any of paragraphs 1-31, which is a hybrid orchimeric polypeptide in which the carbohydrate binding domain of thevariant is replaced with a different carbohydrate binding domain.

[40] The variant of any of paragraphs 1-31, wherein the parent is afusion protein in which a heterologous carbohydrate binding domain isfused to the parent.

[41] The variant of paragraph 40, wherein the carbohydrate bindingdomain is fused to the N-terminus or the C-terminus of the parent.

[42] The variant of paragraph 40 or 41, wherein the fusion proteincomprises or consists of SEQ ID NO: 73 or the mature polypeptidethereof.

[43] The variant of any of paragraphs 1-31, which is a fusion protein inwhich a heterologous carbohydrate binding domain is fused to thevariant.

[44] The variant of paragraph 43, wherein the carbohydrate bindingdomain is fused to the N-terminus or the C-terminus of the variant.

[45] The variant of paragraph 43 or 44, which comprises or consists ofSEQ ID NO: 76 or the mature polypeptide thereof.

[46] The variant of any of paragraphs 1-45, which has an increasedspecific performance relative to the parent.

[47] An isolated polynucleotide encoding the variant of any ofparagraphs 1-46.

[48] A nucleic acid construct or expression vector comprising thepolynucleotide of paragraph 47.

[49] A host cell comprising the polynucleotide of paragraph 47.

[50] A method of producing a cellobiohydrolase variant, comprising:cultivating the host cell of paragraph 49 under conditions suitable forexpression of the variant.

[51] The method of paragraph 50, further comprising recovering thevariant.

[52] A transgenic plant, plant part or plant cell transformed with thepolynucleotide of paragraph 47.

[53] A method of producing the variant of any of paragraphs 1-46,comprising: cultivating a transgenic plant or a plant cell comprising apolynucleotide encoding the variant under conditions conducive forproduction of the variant.

[54] The method of paragraph 53, further comprising recovering thevariant

[55] A method for obtaining a cellobiohydrolase variant, comprisingintroducing into a parent cellobiohydrolase an alteration at one or morepositions corresponding to positions 197, 198, 199, and 200 of themature polypeptide of SEQ ID NO: 2, wherein the alteration at the one ormore positions corresponding to positions 197, 198, and 200 is asubstitution and the alteration at the position corresponding toposition 199 is a deletion, and wherein the variant hascellobiohydrolase activity.

[56] The method of paragraph 55, further comprising recovering thevariant.

[57] A composition comprising the variant of any of paragraphs 1-46.

[58] A whole broth formulation or cell culture composition comprisingthe variant of any of paragraphs 1-46.

[59] A process for degrading a cellulosic material, comprising: treatingthe cellulosic material with an enzyme composition in the presence ofthe variant of any of paragraphs 1-46.

[60] The process of paragraph 59, wherein the cellulosic material ispretreated.

[61] The process of paragraph 59 or 60, wherein the enzyme compositioncomprises one or more enzymes selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, a catalase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

[62] The process of paragraph 61, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[63] The process of paragraph 61, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[64] The process of any of paragraphs 59-63, further comprisingrecovering the degraded cellulosic material.

[65] The process of paragraph 64, wherein the degraded cellulosicmaterial is a sugar.

[66] The process of paragraph 65, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[67] A process for producing a fermentation product, comprising: (a)saccharifying a cellulosic material with an enzyme composition in thepresence of the variant of any of paragraphs 1-46; (b) fermenting thesaccharified cellulosic material with one or more fermentingmicroorganisms to produce the fermentation product; and (c) recoveringthe fermentation product from the fermentation.

[68] The process of paragraph 67, wherein the cellulosic material ispretreated.

[69] The process of paragraph 67 or 68, wherein the enzyme compositioncomprises the enzyme composition comprises one or more enzymes selectedfrom the group consisting of a cellulase, a GH61 polypeptide havingcellulolytic enhancing activity, a hemicellulase, a catalase, anesterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, aperoxidase, a protease, and a swollenin.

[70] The process of paragraph 69, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[71] The process of paragraph 69, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[72] The process of any of paragraphs 67-71, wherein steps (a) and (b)are performed simultaneously in a simultaneous saccharification andfermentation.

[73] The process of any of paragraphs 67-72, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

[74] A process of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more fermentingmicroorganisms, wherein the cellulosic material is saccharified with anenzyme composition in the presence of the variant of any of paragraphs1-46.

[75] The process of paragraph 74, wherein the fermenting of thecellulosic material produces a fermentation product.

[76] The process of paragraph 75, further comprising recovering thefermentation product from the fermentation.

[77] The process of paragraph 75 or 76, wherein the fermentation productis an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, agas, isoprene, a ketone, an organic acid, or polyketide.

[78] The process of any of paragraphs 74-77, wherein the cellulosicmaterial is pretreated before saccharification.

[79] The process of any of paragraphs 74-78, wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a GH61 polypeptide having cellulolyticenhancing activity, a hemicellulase, a catalase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

[80] The process of paragraph 79, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[81] The process of paragraph 80, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

The inventions described and claimed herein are not to be limited inscope by the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the inventions. Anyequivalent aspects are intended to be within the scope of theinventions. Indeed, various modifications of the inventions in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description. Such modificationsare also intended to fall within the scope of the appended claims. Inthe case of conflict, the present disclosure including definitions willcontrol.

What is claimed is:
 1. An isolated polynucleotide encoding acellobiohydrolase I variant, wherein the cellobiohydrolase I variant hascellobiohydrolase I activity and increased specific performance relativeto a cellobiohydrolase I comprising the amino acid sequence of aminoacids 1 to 497 of SEQ ID NO: 2, wherein the cellobiohydrolase I variantcomprises an amino acid sequence having at least 90% sequence identityto the amino acid sequence of amino acids 1 to 497 of SEQ ID NO: 2, andwherein the Asn at the position corresponding to position 197 of theamino acid sequence of amino acids 1 to 497 of SEQ ID NO: 2 issubstituted with Ala in the cellobiohydrolase I variant, the Ala at theposition corresponding to position 199 of the amino acid sequence ofamino acids 1 to 497 of SEQ ID NO: 2 is deleted in the cellobiohydrolaseI variant, and the Asn at the position corresponding to position 200 ofthe amino acid sequence of amino acids 1 to 497 of SEQ ID NO: 2 issubstituted with Ala or Trp in the cellobiohydrolase I variant.
 2. Thepolynucleotide of claim 1, wherein the Asn at the position correspondingto position 198 of the amino acid sequence of amino acids 1 to 497 ofSEQ ID NO: 2 is substituted with Ala in the cellobiohydrolase I variant.3. The polynucleotide of claim 1, wherein the amino acid sequence of thecellobiohydrolase I variant has at least 91% sequence identity to theamino acid sequence of amino acids 1 to 497 of SEQ ID NO:
 2. 4. Thepolynucleotide of claim 1, wherein the amino acid sequence of thecellobiohydrolase I variant has at least 92% sequence identity to theamino acid sequence of amino acids 1 to 497 of SEQ ID NO:
 2. 5. Thepolynucleotide of claim 1, wherein the amino acid sequence of thecellobiohydrolase I variant has at least 93% sequence identity to theamino acid sequence of amino acids 1 to 497 of SEQ ID NO:
 2. 6. Thepolynucleotide of claim 1, wherein the amino acid sequence of thecellobiohydrolase I variant has at least 94% sequence identity to theamino acid sequence of amino acids 1 to 497 of SEQ ID NO:
 2. 7. Thepolynucleotide of claim 1, wherein the amino acid sequence of thecellobiohydrolase I variant has at least 95% sequence identity to theamino acid sequence of amino acids 1 to 497 of SEQ ID NO:
 2. 8. Thepolynucleotide of claim 1, wherein the amino acid sequence of thecellobiohydrolase I variant has at least 96% sequence identity to theamino acid sequence of amino acids 1 to 497 of SEQ ID NO:
 2. 9. Thepolynucleotide of claim 1, wherein the amino acid sequence of thecellobiohydrolase I variant has at least 97% sequence identity to theamino acid sequence of amino acids 1 to 497 of SEQ ID NO:
 2. 10. Thepolynucleotide of claim 1, wherein the amino acid sequence of thecellobiohydrolase I variant has at least 98% sequence identity to theamino acid sequence of amino acids 1 to 497 of SEQ ID NO:
 2. 11. Thepolynucleotide of claim 1, wherein the amino acid sequence of thecellobiohydrolase I variant has at least 99% sequence identity to theamino acid sequence of amino acids 1 to 497 of SEQ ID NO:
 2. 12. Anucleic acid construct comprising the polynucleotide of claim
 1. 13. Anexpression vector comprising the nucleic acid construct of claim
 12. 14.An isolated recombinant host cell transformed with the expression vectorof claim
 13. 15. A method of producing a cellobiohydrolase I variant,comprising: (a) cultivating the recombinant host cell of claim 14 underconditions for production of the cellobiohydrolase I variant, andoptionally (b) recovering the cellobiohydrolase I variant.
 16. Therecombinant host cell of claim 14, wherein the recombinant host cell isa plant cell.